A trichomoniasis vaccine

Abstract

The present disclosure relates to a novel isolated protozoal strain of the species Tritrichomonas foetus. More particularly, this disclosure relates to the development of a vaccine for treating, preventing, or ameliorating infections caused by this protozoal species.

Description

“A Trichomoniasis Vaccine” Related Application Data

[0001] The present application claims priority from Australian Patent Application No. 2024901295 filed on 6 May 2024 entitled “A Trichomoniasis Vaccine”. The entire contents of which is hereby incorporated by reference. Sequence Listing

[0002] The present application is filed with a Sequence Listing, which has been submitted electronically in XML format form and is hereby incorporated by reference in its entirety. Said XML copy, created on 4thApril 2024, is named “TfOz5_masked.fasta” and is 107 Mb in size. Technical Field

[0003] The present disclosure relates to a novel isolated protozoal strain of the species Tritrichomonas foetus. More particularly, this disclosure relates to a vaccine for treating, preventing or ameliorating a protozoal infection. Background

[0004] Bovine trichomonosis or trichomoniasis caused by Tritrichomonas foetus is a sexually transmitted disease that is included in the list of diseases of the World Organization for Animal Health (OIE). Bovine trichomonosis is a primary cause of early reproductive failure in cattle, and can lead to significant financial losses. The disease is associated with lowered fertility in infected herds and a spread out calving season, resulting in a significant negative impact on the productivity in beef cattle herds.

[0005] The parasite is localized in the preputial cavity of bulls and the genital tract of cows, and it is transmitted during coitus. T. foetus infection is asymptomatic in males, and it does not affect semen quality or sexual behaviour. However, the bull is a lifelong asymptomatic carrier and transmitter of the disease. Parasite multiplication causes inflammation of the reproductive tract, including cervicitis and endometritis, in cows or heifers following infection at mating and resulting in embryonic death or early abortion. While infection in females is generally self-limiting and resolves after two to four months, the immunity that develops is not permanent and generally only lasts for approximately six months; then the female is susceptible to re-infection.

[0006] Although numerous studies have been carried out, there are only two inactivated vaccines against bovine trichomonosis available on the market: TrichGuard (Boehringer Ingelheim) and Tricovac (Laboratorio Biológico, Tandil) commercialized in America and Argentina, respectively. Furthermore, these vaccines have not been shown to be able to induce protection in bulls (indeed the recommendation is to vaccinate all breeding females), rather only being able to reduce the clearance time of T. foetus from the reproductive tract before foetal loss occurs and improvepregnancy rates by decreasing the duration of endometritis (Herr et al.1991; Kvasnicka et al.1989; Kvasnicka et al.1992; Hudson et al.1993; Edmondson et al.2017; Fuchs et al.2017).

[0007] In Australia, there is evidence of a re-emergence of bovine trichomonosis in husbandry conditions (McGowan et al.2021; Irons et al.2022). Accordingly, there remains a clinical need for a vaccine to prevent and treat the effects of T. foetus in Australian cattle, and particularly in bulls, which improves on the efficacy of commercial vaccines. Summary

[0008] The present disclosure is based on the identification of conserved protozoal strains of the species Tritrichomonas foetus. The inventors were able to isolate and sequence these novel protozoal strains of the species Tritrichomonas foetus from Australian cattle herds. Surprisingly, the inventors also found that an immunogenic composition comprising a protozoal strain of the species Tritrichomonas foetus shows an immunogenic effect and the prevention or clearance of infection with this protozoan in cattle, and more particularly bulls.

[0009] In a first aspect, the present disclosure relates to an immunogenic composition comprising a protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

[0010] In a second aspect, the present disclosure relates to an isolated Tritrichomonas foetus strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

[0011] In a third aspect, the present disclosure relates to an isolated cell or isolated population of cells of a Tritrichomonas foetus strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

[0012] In particular examples of the above aspects, the genomic sequence of the Tritrichomonas foetus strain comprises or is assembled from nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof.

[0013] According to other examples of the above aspects, the genomic sequence of the Tritrichomonas foetus strain comprises or is assembled from nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof.

[0014] According to other examples of the above aspects, the genomic sequence of the Tritrichomonas foetus strain comprises or is assembled from nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

[0015] In a fourth aspect, the present disclosure relates to a method of producing the immunogenic composition of the first aspect, said method including the step of culturing one or more cells of the protozoal strain in a cell culture medium.

[0016] Referring to the fourth aspect, the method further includes the step of at least partially isolating the one or more cells from the cell culture medium.

[0017] Suitably, the method further including the step of at least partly inactivating or attenuating the one or more cells of the protozoal strain.

[0018] In some examples, the cell culture medium is or comprises of Tritrichomonas foetus medium (TFM). In one example, the TFM contains no agar or is substantially free of agar.

[0019] In particular examples, the cell culture medium is a liquid cell culture medium.

[0020] In certain examples, the cell culture medium further comprises at least one antimicrobial agent selected from the group consisting of an antibacterial, an antifungal, an antimycotic and combinations thereof. Suitably, the at least one antimicrobial agent is selected from the group consisting of amphotericin B, penicillin, streptomycin, gentamicin, tetracycline, normacin, nystatin and combinations thereof.

[0021] In a fifth aspect, the present disclosure relates to a method of eliciting an immune response in a mammal, the method comprising administering a first dose comprising a therapeutically effective amount of the immunogenic composition of the first aspect to the mammal to thereby elicit an immune response.

[0022] In a sixth aspect, the present disclosure relates to use of an immunogenic composition of the first aspect in the manufacture of a medicament for eliciting an immune response in a mammal.

[0023] In a seventh aspect, the present disclosure relates to an immunogenic composition of the first aspect for use in eliciting an immune response in a mammal.

[0024] In an eighth aspect, the present disclosure relates to a method of treating, preventing or ameliorating a protozoal infection in a mammal, the method comprising administering a first dose comprising a therapeutically effective amount of the immunogenic composition of the first aspect to the mammal to thereby treat, prevent or ameliorate the protozoal infection.

[0025] In a ninth aspect, the present disclosure relates to the use of an immunogenic composition of the first aspect in the manufacture of a medicament for treating, preventing or ameliorating a protozoal infection in a mammal.

[0026] In a tenth aspect, the present disclosure relates to an immunogenic composition of the first aspect for use treating, preventing or ameliorating a protozoal infection in a mammal.

[0027] Referring to the fifth, seventh, eighth and tenth aspects, the method suitably further includes the step of administering a second dose comprising a therapeutically effective amount of the immunogenic composition of the first aspect after the first dose. Similarly, referring to the sixth and ninth aspects, the medicament is formulated for administration as a first dose prior to administration of a second dose. Alternatively, the medicament may be formulated for administration as a second dose after administration of a first dose.

[0028] In some examples, the second dose is administered to the mammal at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days after administration of the first dose. In particular, the medicament is formulated to be administered prior to a second dose.

[0029] Referring to the fifth, seventh, eighth and tenth aspects the immunogenic composition is suitably administered parenterally. Similarly, referring to the sixth and ninth aspects, the medicament is suitably formulated for parenteral administration.

[0030] Suitably, the immunogenic composition comprises one or more pharmaceutically acceptable carriers, diluents or excipients.

[0031] For the aforementioned aspects, the protozoal strain can have a genomic sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194.

[0032] In various examples, the protozoal strain can have a genomic sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 195 to 562.

[0033] In various examples, the protozoal strain can have a genomic sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 563 to 756.

[0034] In particular examples, the protozoal strain has the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194.

[0035] In other examples, the protozoal strain has the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 195 to 562.

[0036] In other examples, the protozoal strain has the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 563 to 756.

[0037] In some examples, the protozoal strain is a Tritrichomonas foetus strain Oz5 or a Tritrichomonas foetus strain Oz-N36. For example, a Tritrichomonas foetus strain Oz5 of ordeposited under ATCC Accession Number PTA-127585. In another example, a Tritrichomonas foetus strain Oz-N36 of or deposited under ATCC Accession Number PTA-127586.

[0038] In various examples, the protozoal strain is or has been inactivated or attenuated.

[0039] In some examples, the protozoal strain is or has been inactivated or attenuated by any one of pasteurisation, an acidic pH, a solvent / detergent treatment, ultraviolet light and combinations thereof.

[0040] In particular examples, the immunogenic composition comprises about 1 x 104to about 1 x 109cells of the protozoal strain per unit dose.

[0041] Suitably, the immunogenic composition comprises an adjuvant. For example, the adjuvant is or comprises a water-in-oil emulsion. In particular examples, the adjuvant is a Montanide adjuvant.

[0042] The present disclosure also provides a protozoal strain produced by the methods described herein, such as that of the fourth aspect. Brief description of the drawings

[0043] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific examples presented herein. It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described examples, without departing from the broad general scope of the present disclosure. The present examples are, therefore, to be considered in all respects as illustrative and not restrictive.

[0044] Figure 1: Gel Electrophoresis 0.8% TBE-Agarose 80V for 60 minutes using SYBR Safe stain. (A) T. foetus degraded genomic DNA resulting from different extraction methods: (1) YVL-W strain; (2) TfOz5 isolate; (3) TfOz5 agar fraction (nuclear DNA extractions); (4) YVL-W late subculture; (5) YVL-W early subculture; (6) TfOz5 (standard lysis and phenol chloroform extractions). Ladder: Marker 1kb plus. (B) T. foetus genomic DNA resulting from alkaline / 4°C lysis method / phenol chloroform extraction with (1) YVL-W strain, (2) TfOz5 isolate, and (3) TfOz-N36 isolate. Ladder: Marker 1kb plus.

[0045] Figure 2: Averaged T. foetus IgG responses between vaccinated and un-vaccinated bulls.06 / 06 and 23 / 09 are the dates of challenge 1 and challenge 2 respectively.

[0046] Figure 3: Scheme of the bull vaccine trial (n=60). Blood and preputial samples were collected at multiple time points throughout the trial as indicated by arrows, ending on Day 189.

[0047] Figure 4: Average length of T. foetus infection in the genital tract. Data from each animal are presented as individual points. Square=control. Circle=vaccine. Horizontal linesrepresent median values for each group. Data were analysed using two-tailed Mann-Whitney test to compare vaccine vs. control group. **p < 0.01.

[0048] Figure 5: Numbers of the T. foetus infected bulls in the vaccinated group and the non- immunised group (Control). First broken line indicates the second immunisation (Day 27), second broken line indicates first challenge and third broken line indicates second challenge with T. foetus. Key to the Sequence Listing

[0049] The following table provides SEQ ID NOs and names that correspond to the names presented in the sequence listing provided herein. SEQ ID NO: 1 Nucleotide sequence of contig_1004 of TfOz5 SEQ ID NO: 2 Nucleotide sequence of contig_1008 of TfOz5 SEQ ID NO: 3 Nucleotide sequence of contig_1022 of TfOz5 SEQ ID NO: 4 Nucleotide sequence of contig_1028 of TfOz5 SEQ ID NO: 5 Nucleotide sequence of contig_1037 of TfOz5 SEQ ID NO: 6 Nucleotide sequence of contig_1041 of TfOz5 SEQ ID NO: 7 Nucleotide sequence of contig_1049 of TfOz5 SEQ ID NO: 8 Nucleotide sequence of contig_1053 of TfOz5 SEQ ID NO: 9 Nucleotide sequence of contig_1069 of TfOz5 SEQ ID NO: 10 Nucleotide sequence of contig_1071 of TfOz5 SEQ ID NO: 11 Nucleotide sequence of contig_1072 of TfOz5 SEQ ID NO: 12 Nucleotide sequence of contig_1074 of TfOz5 SEQ ID NO: 13 Nucleotide sequence of contig_1078 of TfOz5 SEQ ID NO: 14 Nucleotide sequence of contig_1086 of TfOz5 SEQ ID NO: 15 Nucleotide sequence of contig_1088 of TfOz5 SEQ ID NO: 16 Nucleotide sequence of contig_1106 of TfOz5 SEQ ID NO: 17 Nucleotide sequence of contig_112 of TfOz5 SEQ ID NO: 18 Nucleotide sequence of contig_1122 of TfOz5 SEQ ID NO: 19 Nucleotide sequence of contig_1124 of TfOz5 SEQ ID NO: 20 Nucleotide sequence of contig_1125 of TfOz5 SEQ ID NO: 21 Nucleotide sequence of contig_1139 of TfOz5 SEQ ID NO: 22 Nucleotide sequence of contig_1141 of TfOz5 SEQ ID NO: 23 Nucleotide sequence of contig_1158 of TfOz5 SEQ ID NO: 24 Nucleotide sequence of contig_1164 of TfOz5 SEQ ID NO: 25 Nucleotide sequence of contig_1165 of TfOz5 SEQ ID NO: 26 Nucleotide sequence of contig_1180 of TfOz5 SEQ ID NO: 27 Nucleotide sequence of contig_1184 of TfOz5 SEQ ID NO: 28 Nucleotide sequence of contig_1194 of TfOz5 SEQ ID NO: 29 Nucleotide sequence of contig_1206 of TfOz5 SEQ ID NO: 30 Nucleotide sequence of contig_1207 of TfOz5 SEQ ID NO: 31 Nucleotide sequence of contig_1215 of TfOz5 SEQ ID NO: 32 Nucleotide sequence of contig_1217 of TfOz5 SEQ ID NO: 33 Nucleotide sequence of contig_1225 of TfOz5 SEQ ID NO: 34 Nucleotide sequence of contig_1232 of TfOz5SEQ ID NO: 35 Nucleotide sequence of contig_1244 of TfOz5 SEQ ID NO: 36 Nucleotide sequence of contig_1246 of TfOz5 SEQ ID NO: 37 Nucleotide sequence of contig_1263 of TfOz5 SEQ ID NO: 38 Nucleotide sequence of contig_1266 of TfOz5 SEQ ID NO: 39 Nucleotide sequence of contig_1267 of TfOz5 SEQ ID NO: 40 Nucleotide sequence of contig_1270 of TfOz5 SEQ ID NO: 41 Nucleotide sequence of contig_1279 of TfOz5 SEQ ID NO: 42 Nucleotide sequence of contig_1281 of TfOz5 SEQ ID NO: 43 Nucleotide sequence of contig_129 of TfOz5 SEQ ID NO: 44 Nucleotide sequence of contig_1300 of TfOz5 SEQ ID NO: 45 Nucleotide sequence of contig_1319 of TfOz5 SEQ ID NO: 46 Nucleotide sequence of contig_1346 of TfOz5 SEQ ID NO: 47 Nucleotide sequence of contig_1350 of TfOz5 SEQ ID NO: 48 Nucleotide sequence of contig_1364 of TfOz5 SEQ ID NO: 49 Nucleotide sequence of contig_1366 of TfOz5 SEQ ID NO: 50 Nucleotide sequence of contig_138 of TfOz5 SEQ ID NO: 51 Nucleotide sequence of contig_141 of TfOz5 SEQ ID NO: 52 Nucleotide sequence of contig_143 of TfOz5 SEQ ID NO: 53 Nucleotide sequence of contig_153 of TfOz5 SEQ ID NO: 54 Nucleotide sequence of contig_17 of TfOz5 SEQ ID NO: 55 Nucleotide sequence of contig_173 of TfOz5 SEQ ID NO: 56 Nucleotide sequence of contig_176 of TfOz5 SEQ ID NO: 57 Nucleotide sequence of contig_178 of TfOz5 SEQ ID NO: 58 Nucleotide sequence of contig_179 of TfOz5 SEQ ID NO: 59 Nucleotide sequence of contig_183 of TfOz5 SEQ ID NO: 60 Nucleotide sequence of contig_187 of TfOz5 SEQ ID NO: 61 Nucleotide sequence of contig_194 of TfOz5 SEQ ID NO: 62 Nucleotide sequence of contig_199 of TfOz5 SEQ ID NO: 63 Nucleotide sequence of contig_2 of TfOz5 SEQ ID NO: 64 Nucleotide sequence of contig_204 of TfOz5 SEQ ID NO: 65 Nucleotide sequence of contig_210 of TfOz5 SEQ ID NO: 66 Nucleotide sequence of contig_214 of TfOz5 SEQ ID NO: 67 Nucleotide sequence of contig_217 of TfOz5 SEQ ID NO: 68 Nucleotide sequence of contig_220 of TfOz5 SEQ ID NO: 69 Nucleotide sequence of contig_224 of TfOz5 SEQ ID NO: 70 Nucleotide sequence of contig_237 of TfOz5 SEQ ID NO: 71 Nucleotide sequence of contig_245 of TfOz5 SEQ ID NO: 72 Nucleotide sequence of contig_246 of TfOz5 SEQ ID NO: 73 Nucleotide sequence of contig_252 of TfOz5 SEQ ID NO: 74 Nucleotide sequence of contig_262 of TfOz5 SEQ ID NO: 75 Nucleotide sequence of contig_266 of TfOz5 SEQ ID NO: 76 Nucleotide sequence of contig_268 of TfOz5 SEQ ID NO: 77 Nucleotide sequence of contig_270 of TfOz5 SEQ ID NO: 78 Nucleotide sequence of contig_276 of TfOz5 SEQ ID NO: 79 Nucleotide sequence of contig_281 of TfOz5 SEQ ID NO: 80 Nucleotide sequence of contig_285 of TfOz5 SEQ ID NO: 81 Nucleotide sequence of contig_286 of TfOz5SEQ ID NO: 82 Nucleotide sequence of contig_288 of TfOz5 SEQ ID NO: 83 Nucleotide sequence of contig_291 of TfOz5 SEQ ID NO: 84 Nucleotide sequence of contig_294 of TfOz5 SEQ ID NO: 85 Nucleotide sequence of contig_296 of TfOz5 SEQ ID NO: 86 Nucleotide sequence of contig_302 of TfOz5 SEQ ID NO: 87 Nucleotide sequence of contig_303 of TfOz5 SEQ ID NO: 88 Nucleotide sequence of contig_317 of TfOz5 SEQ ID NO: 89 Nucleotide sequence of contig_320 of TfOz5 SEQ ID NO: 90 Nucleotide sequence of contig_330 of TfOz5 SEQ ID NO: 91 Nucleotide sequence of contig_341 of TfOz5 SEQ ID NO: 92 Nucleotide sequence of contig_342 of TfOz5 SEQ ID NO: 93 Nucleotide sequence of contig_347 of TfOz5 SEQ ID NO: 94 Nucleotide sequence of contig_356 of TfOz5 SEQ ID NO: 95 Nucleotide sequence of contig_366 of TfOz5 SEQ ID NO: 96 Nucleotide sequence of contig_369 of TfOz5 SEQ ID NO: 97 Nucleotide sequence of contig_372 of TfOz5 SEQ ID NO: 98 Nucleotide sequence of contig_377 of TfOz5 SEQ ID NO: 99 Nucleotide sequence of contig_381 of TfOz5 SEQ ID NO: 100 Nucleotide sequence of contig_386 of TfOz5 SEQ ID NO: 101 Nucleotide sequence of contig_392 of TfOz5 SEQ ID NO: 102 Nucleotide sequence of contig_396 of TfOz5 SEQ ID NO: 103 Nucleotide sequence of contig_397 of TfOz5 SEQ ID NO: 104 Nucleotide sequence of contig_398 of TfOz5 SEQ ID NO: 105 Nucleotide sequence of contig_4 of TfOz5 SEQ ID NO: 106 Nucleotide sequence of contig_40 of TfOz5 SEQ ID NO: 107 Nucleotide sequence of contig_402 of TfOz5 SEQ ID NO: 108 Nucleotide sequence of contig_409 of TfOz5 SEQ ID NO: 109 Nucleotide sequence of contig_411 of TfOz5 SEQ ID NO: 110 Nucleotide sequence of contig_418 of TfOz5 SEQ ID NO: 111 Nucleotide sequence of contig_422 of TfOz5 SEQ ID NO: 112 Nucleotide sequence of contig_427 of TfOz5 SEQ ID NO: 113 Nucleotide sequence of contig_435 of TfOz5 SEQ ID NO: 114 Nucleotide sequence of contig_440 of TfOz5 SEQ ID NO: 115 Nucleotide sequence of contig_446 of TfOz5 SEQ ID NO: 116 Nucleotide sequence of contig_447 of TfOz5 SEQ ID NO: 117 Nucleotide sequence of contig_459 of TfOz5 SEQ ID NO: 118 Nucleotide sequence of contig_471 of TfOz5 SEQ ID NO: 119 Nucleotide sequence of contig_475 of TfOz5 SEQ ID NO: 120 Nucleotide sequence of contig_479 of TfOz5 SEQ ID NO: 121 Nucleotide sequence of contig_482 of TfOz5 SEQ ID NO: 122 Nucleotide sequence of contig_490 of TfOz5 SEQ ID NO: 123 Nucleotide sequence of contig_497 of TfOz5 SEQ ID NO: 124 Nucleotide sequence of contig_499 of TfOz5 SEQ ID NO: 125 Nucleotide sequence of contig_5 of TfOz5 SEQ ID NO: 126 Nucleotide sequence of contig_502 of TfOz5 SEQ ID NO: 127 Nucleotide sequence of contig_521 of TfOz5 SEQ ID NO: 128 Nucleotide sequence of contig_527 of TfOz5SEQ ID NO: 129 Nucleotide sequence of contig_530 of TfOz5 SEQ ID NO: 130 Nucleotide sequence of contig_552 of TfOz5 SEQ ID NO: 131 Nucleotide sequence of contig_559 of TfOz5 SEQ ID NO: 132 Nucleotide sequence of contig_56 of TfOz5 SEQ ID NO: 133 Nucleotide sequence of contig_561 of TfOz5 SEQ ID NO: 134 Nucleotide sequence of contig_565 of TfOz5 SEQ ID NO: 135 Nucleotide sequence of contig_572 of TfOz5 SEQ ID NO: 136 Nucleotide sequence of contig_574 of TfOz5 SEQ ID NO: 137 Nucleotide sequence of contig_577 of TfOz5 SEQ ID NO: 138 Nucleotide sequence of contig_580 of TfOz5 SEQ ID NO: 139 Nucleotide sequence of contig_584 of TfOz5 SEQ ID NO: 140 Nucleotide sequence of contig_594 of TfOz5 SEQ ID NO: 141 Nucleotide sequence of contig_616 of TfOz5 SEQ ID NO: 142 Nucleotide sequence of contig_62 of TfOz5 SEQ ID NO: 143 Nucleotide sequence of contig_625 of TfOz5 SEQ ID NO: 144 Nucleotide sequence of contig_631 of TfOz5 SEQ ID NO: 145 Nucleotide sequence of contig_637 of TfOz5 SEQ ID NO: 146 Nucleotide sequence of contig_646 of TfOz5 SEQ ID NO: 147 Nucleotide sequence of contig_65 of TfOz5 SEQ ID NO: 148 Nucleotide sequence of contig_655 of TfOz5 SEQ ID NO: 149 Nucleotide sequence of contig_659 of TfOz5 SEQ ID NO: 150 Nucleotide sequence of contig_662 of TfOz5 SEQ ID NO: 151 Nucleotide sequence of contig_671 of TfOz5 SEQ ID NO: 152 Nucleotide sequence of contig_672 of TfOz5 SEQ ID NO: 153 Nucleotide sequence of contig_691 of TfOz5 SEQ ID NO: 154 Nucleotide sequence of contig_701 of TfOz5 SEQ ID NO: 155 Nucleotide sequence of contig_709 of TfOz5 SEQ ID NO: 156 Nucleotide sequence of contig_719 of TfOz5 SEQ ID NO: 157 Nucleotide sequence of contig_721 of TfOz5 SEQ ID NO: 158 Nucleotide sequence of contig_723 of TfOz5 SEQ ID NO: 159 Nucleotide sequence of contig_725 of TfOz5 SEQ ID NO: 160 Nucleotide sequence of contig_736 of TfOz5 SEQ ID NO: 161 Nucleotide sequence of contig_737 of TfOz5 SEQ ID NO: 162 Nucleotide sequence of contig_765 of TfOz5 SEQ ID NO: 163 Nucleotide sequence of contig_768 of TfOz5 SEQ ID NO: 164 Nucleotide sequence of contig_775 of TfOz5 SEQ ID NO: 165 Nucleotide sequence of contig_788 of TfOz5 SEQ ID NO: 166 Nucleotide sequence of contig_796 of TfOz5 SEQ ID NO: 167 Nucleotide sequence of contig_799 of TfOz5 SEQ ID NO: 168 Nucleotide sequence of contig_804 of TfOz5 SEQ ID NO: 169 Nucleotide sequence of contig_806 of TfOz5 SEQ ID NO: 170 Nucleotide sequence of contig_81 of TfOz5 SEQ ID NO: 171 Nucleotide sequence of contig_813 of TfOz5 SEQ ID NO: 172 Nucleotide sequence of contig_816 of TfOz5 SEQ ID NO: 173 Nucleotide sequence of contig_821 of TfOz5 SEQ ID NO: 174 Nucleotide sequence of contig_823 of TfOz5 SEQ ID NO: 175 Nucleotide sequence of contig_825 of TfOz5SEQ ID NO: 176 Nucleotide sequence of contig_837 of TfOz5 SEQ ID NO: 177 Nucleotide sequence of contig_841 of TfOz5 SEQ ID NO: 178 Nucleotide sequence of contig_859 of TfOz5 SEQ ID NO: 179 Nucleotide sequence of contig_863 of TfOz5 SEQ ID NO: 180 Nucleotide sequence of contig_874 of TfOz5 SEQ ID NO: 181 Nucleotide sequence of contig_899 of TfOz5 SEQ ID NO: 182 Nucleotide sequence of contig_915 of TfOz5 SEQ ID NO: 183 Nucleotide sequence of contig_924 of TfOz5 SEQ ID NO: 184 Nucleotide sequence of contig_926 of TfOz5 SEQ ID NO: 185 Nucleotide sequence of contig_932 of TfOz5 SEQ ID NO: 186 Nucleotide sequence of contig_938 of TfOz5 SEQ ID NO: 187 Nucleotide sequence of contig_944 of TfOz5 SEQ ID NO: 188 Nucleotide sequence of contig_963 of TfOz5 SEQ ID NO: 189 Nucleotide sequence of contig_966 of TfOz5 SEQ ID NO: 190 Nucleotide sequence of contig_970 of TfOz5 SEQ ID NO: 191 Nucleotide sequence of contig_984 of TfOz5 SEQ ID NO: 192 Nucleotide sequence of contig_987 of TfOz5 SEQ ID NO: 193 Nucleotide sequence of contig_988 of TfOz5 SEQ ID NO: 194 Nucleotide sequence of contig_998 of TfOz5 SEQ ID NO: 195 Nucleotide sequence of contig_1000 of TfOz-N36 SEQ ID NO: 196 Nucleotide sequence of contig_1011 of TfOz-N36 SEQ ID NO: 197 Nucleotide sequence of contig_1014 of TfOz-N36 SEQ ID NO: 198 Nucleotide sequence of contig_1018 of TfOz-N36 SEQ ID NO: 199 Nucleotide sequence of contig_102 of TfOz-N36 SEQ ID NO: 200 Nucleotide sequence of contig_1020 of TfOz-N36 SEQ ID NO: 201 Nucleotide sequence of contig_1021 of TfOz-N36 SEQ ID NO: 202 Nucleotide sequence of contig_1024 of TfOz-N36 SEQ ID NO: 203 Nucleotide sequence of contig_1025 of TfOz-N36 SEQ ID NO: 204 Nucleotide sequence of contig_1034 of TfOz-N36 SEQ ID NO: 205 Nucleotide sequence of contig_1041 of TfOz-N36 SEQ ID NO: 206 Nucleotide sequence of contig_1049 of TfOz-N36 SEQ ID NO: 207 Nucleotide sequence of contig_1055 of TfOz-N36 SEQ ID NO: 208 Nucleotide sequence of contig_1059 of TfOz-N36 SEQ ID NO: 209 Nucleotide sequence of contig_106 of TfOz-N36 SEQ ID NO: 210 Nucleotide sequence of contig_1062 of TfOz-N36 SEQ ID NO: 211 Nucleotide sequence of contig_1065 of TfOz-N36 SEQ ID NO: 212 Nucleotide sequence of contig_1066 of TfOz-N36 SEQ ID NO: 213 Nucleotide sequence of contig_1067 of TfOz-N36 SEQ ID NO: 214 Nucleotide sequence of contig_1068 of TfOz-N36 SEQ ID NO: 215 Nucleotide sequence of contig_107 of TfOz-N36 SEQ ID NO: 216 Nucleotide sequence of contig_1076 of TfOz-N36 SEQ ID NO: 217 Nucleotide sequence of contig_1082 of TfOz-N36 SEQ ID NO: 218 Nucleotide sequence of contig_1092 of TfOz-N36 SEQ ID NO: 219 Nucleotide sequence of contig_1098 of TfOz-N36 SEQ ID NO: 220 Nucleotide sequence of contig_110 of TfOz-N36 SEQ ID NO: 221 Nucleotide sequence of contig_1108 of TfOz-N36 SEQ ID NO: 222 Nucleotide sequence of contig_1110 of TfOz-N36SEQ ID NO: 223 Nucleotide sequence of contig_1114 of TfOz-N36 SEQ ID NO: 224 Nucleotide sequence of contig_1115 of TfOz-N36 SEQ ID NO: 225 Nucleotide sequence of contig_1117 of TfOz-N36 SEQ ID NO: 226 Nucleotide sequence of contig_1119 of TfOz-N36 SEQ ID NO: 227 Nucleotide sequence of contig_112 of TfOz-N36 SEQ ID NO: 228 Nucleotide sequence of contig_1121 of TfOz-N36 SEQ ID NO: 229 Nucleotide sequence of contig_1124 of TfOz-N36 SEQ ID NO: 230 Nucleotide sequence of contig_1126 of TfOz-N36 SEQ ID NO: 231 Nucleotide sequence of contig_1127 of TfOz-N36 SEQ ID NO: 232 Nucleotide sequence of contig_1135 of TfOz-N36 SEQ ID NO: 233 Nucleotide sequence of contig_1139 of TfOz-N36 SEQ ID NO: 234 Nucleotide sequence of contig_1141 of TfOz-N36 SEQ ID NO: 235 Nucleotide sequence of contig_1143 of TfOz-N36 SEQ ID NO: 236 Nucleotide sequence of contig_1144 of TfOz-N36 SEQ ID NO: 237 Nucleotide sequence of contig_1147 of TfOz-N36 SEQ ID NO: 238 Nucleotide sequence of contig_1152 of TfOz-N36 SEQ ID NO: 239 Nucleotide sequence of contig_1155 of TfOz-N36 SEQ ID NO: 240 Nucleotide sequence of contig_1158 of TfOz-N36 SEQ ID NO: 241 Nucleotide sequence of contig_1159 of TfOz-N36 SEQ ID NO: 242 Nucleotide sequence of contig_1161 of TfOz-N36 SEQ ID NO: 243 Nucleotide sequence of contig_1162 of TfOz-N36 SEQ ID NO: 244 Nucleotide sequence of contig_1164 of TfOz-N36 SEQ ID NO: 245 Nucleotide sequence of contig_1174 of TfOz-N36 SEQ ID NO: 246 Nucleotide sequence of contig_1179 of TfOz-N36 SEQ ID NO: 247 Nucleotide sequence of contig_1180 of TfOz-N36 SEQ ID NO: 248 Nucleotide sequence of contig_1183 of TfOz-N36 SEQ ID NO: 249 Nucleotide sequence of contig_1189 of TfOz-N36 SEQ ID NO: 250 Nucleotide sequence of contig_1191 of TfOz-N36 SEQ ID NO: 251 Nucleotide sequence of contig_1193 of TfOz-N36 SEQ ID NO: 252 Nucleotide sequence of contig_12 of TfOz-N36 SEQ ID NO: 253 Nucleotide sequence of contig_1204 of TfOz-N36 SEQ ID NO: 254 Nucleotide sequence of contig_1207 of TfOz-N36 SEQ ID NO: 255 Nucleotide sequence of contig_121 of TfOz-N36 SEQ ID NO: 256 Nucleotide sequence of contig_1212 of TfOz-N36 SEQ ID NO: 257 Nucleotide sequence of contig_1215 of TfOz-N36 SEQ ID NO: 258 Nucleotide sequence of contig_1219 of TfOz-N36 SEQ ID NO: 259 Nucleotide sequence of contig_1224 of TfOz-N36 SEQ ID NO: 260 Nucleotide sequence of contig_1226 of TfOz-N36 SEQ ID NO: 261 Nucleotide sequence of contig_1228 of TfOz-N36 SEQ ID NO: 262 Nucleotide sequence of contig_1229 of TfOz-N36 SEQ ID NO: 263 Nucleotide sequence of contig_123 of TfOz-N36 SEQ ID NO: 264 Nucleotide sequence of contig_1231 of TfOz-N36 SEQ ID NO: 265 Nucleotide sequence of contig_1233 of TfOz-N36 SEQ ID NO: 266 Nucleotide sequence of contig_1236 of TfOz-N36 SEQ ID NO: 267 Nucleotide sequence of contig_1239 of TfOz-N36 SEQ ID NO: 268 Nucleotide sequence of contig_124 of TfOz-N36 SEQ ID NO: 269 Nucleotide sequence of contig_1241 of TfOz-N36SEQ ID NO: 270 Nucleotide sequence of contig_1244 of TfOz-N36 SEQ ID NO: 271 Nucleotide sequence of contig_1245 of TfOz-N36 SEQ ID NO: 272 Nucleotide sequence of contig_1257 of TfOz-N36 SEQ ID NO: 273 Nucleotide sequence of contig_126 of TfOz-N36 SEQ ID NO: 274 Nucleotide sequence of contig_1262 of TfOz-N36 SEQ ID NO: 275 Nucleotide sequence of contig_1264 of TfOz-N36 SEQ ID NO: 276 Nucleotide sequence of contig_1281 of TfOz-N36 SEQ ID NO: 277 Nucleotide sequence of contig_1291 of TfOz-N36 SEQ ID NO: 278 Nucleotide sequence of contig_1292 of TfOz-N36 SEQ ID NO: 279 Nucleotide sequence of contig_1295 of TfOz-N36 SEQ ID NO: 280 Nucleotide sequence of contig_1298 of TfOz-N36 SEQ ID NO: 281 Nucleotide sequence of contig_1308 of TfOz-N36 SEQ ID NO: 282 Nucleotide sequence of contig_1309 of TfOz-N36 SEQ ID NO: 283 Nucleotide sequence of contig_1310 of TfOz-N36 SEQ ID NO: 284 Nucleotide sequence of contig_1316 of TfOz-N36 SEQ ID NO: 285 Nucleotide sequence of contig_1317 of TfOz-N36 SEQ ID NO: 286 Nucleotide sequence of contig_1319 of TfOz-N36 SEQ ID NO: 287 Nucleotide sequence of contig_1328 of TfOz-N36 SEQ ID NO: 288 Nucleotide sequence of contig_1330 of TfOz-N36 SEQ ID NO: 289 Nucleotide sequence of contig_1331 of TfOz-N36 SEQ ID NO: 290 Nucleotide sequence of contig_1333 of TfOz-N36 SEQ ID NO: 291 Nucleotide sequence of contig_1340 of TfOz-N36 SEQ ID NO: 292 Nucleotide sequence of contig_1342 of TfOz-N36 SEQ ID NO: 293 Nucleotide sequence of contig_1346 of TfOz-N36 SEQ ID NO: 294 Nucleotide sequence of contig_135 of TfOz-N36 SEQ ID NO: 295 Nucleotide sequence of contig_136 of TfOz-N36 SEQ ID NO: 296 Nucleotide sequence of contig_1360 of TfOz-N36 SEQ ID NO: 297 Nucleotide sequence of contig_1361 of TfOz-N36 SEQ ID NO: 298 Nucleotide sequence of contig_1363 of TfOz-N36 SEQ ID NO: 299 Nucleotide sequence of contig_1366 of TfOz-N36 SEQ ID NO: 300 Nucleotide sequence of contig_1367 of TfOz-N36 SEQ ID NO: 301 Nucleotide sequence of contig_137 of TfOz-N36 SEQ ID NO: 302 Nucleotide sequence of contig_1372 of TfOz-N36 SEQ ID NO: 303 Nucleotide sequence of contig_138 of TfOz-N36 SEQ ID NO: 304 Nucleotide sequence of contig_1380 of TfOz-N36 SEQ ID NO: 305 Nucleotide sequence of contig_1381 of TfOz-N36 SEQ ID NO: 306 Nucleotide sequence of contig_1384 of TfOz-N36 SEQ ID NO: 307 Nucleotide sequence of contig_1385 of TfOz-N36 SEQ ID NO: 308 Nucleotide sequence of contig_1389 of TfOz-N36 SEQ ID NO: 309 Nucleotide sequence of contig_1397 of TfOz-N36 SEQ ID NO: 310 Nucleotide sequence of contig_14 of TfOz-N36 SEQ ID NO: 311 Nucleotide sequence of contig_1400 of TfOz-N36 SEQ ID NO: 312 Nucleotide sequence of contig_1401 of TfOz-N36 SEQ ID NO: 313 Nucleotide sequence of contig_1402 of TfOz-N36 SEQ ID NO: 314 Nucleotide sequence of contig_1404 of TfOz-N36 SEQ ID NO: 315 Nucleotide sequence of contig_141 of TfOz-N36 SEQ ID NO: 316 Nucleotide sequence of contig_1412 of TfOz-N36SEQ ID NO: 317 Nucleotide sequence of contig_1415 of TfOz-N36 SEQ ID NO: 318 Nucleotide sequence of contig_1417 of TfOz-N36 SEQ ID NO: 319 Nucleotide sequence of contig_1427 of TfOz-N36 SEQ ID NO: 320 Nucleotide sequence of contig_1434 of TfOz-N36 SEQ ID NO: 321 Nucleotide sequence of contig_1447 of TfOz-N36 SEQ ID NO: 322 Nucleotide sequence of contig_1470 of TfOz-N36 SEQ ID NO: 323 Nucleotide sequence of contig_1477 of TfOz-N36 SEQ ID NO: 324 Nucleotide sequence of contig_1479 of TfOz-N36 SEQ ID NO: 325 Nucleotide sequence of contig_148 of TfOz-N36 SEQ ID NO: 326 Nucleotide sequence of contig_1481 of TfOz-N36 SEQ ID NO: 327 Nucleotide sequence of contig_1482 of TfOz-N36 SEQ ID NO: 328 Nucleotide sequence of contig_1483 of TfOz-N36 SEQ ID NO: 329 Nucleotide sequence of contig_1485 of TfOz-N36 SEQ ID NO: 330 Nucleotide sequence of contig_1487 of TfOz-N36 SEQ ID NO: 331 Nucleotide sequence of contig_1488 of TfOz-N36 SEQ ID NO: 332 Nucleotide sequence of contig_1489 of TfOz-N36 SEQ ID NO: 333 Nucleotide sequence of contig_1490 of TfOz-N36 SEQ ID NO: 334 Nucleotide sequence of contig_1491 of TfOz-N36 SEQ ID NO: 335 Nucleotide sequence of contig_1492 of TfOz-N36 SEQ ID NO: 336 Nucleotide sequence of contig_150 of TfOz-N36 SEQ ID NO: 337 Nucleotide sequence of contig_151 of TfOz-N36 SEQ ID NO: 338 Nucleotide sequence of contig_156 of TfOz-N36 SEQ ID NO: 339 Nucleotide sequence of contig_158 of TfOz-N36 SEQ ID NO: 340 Nucleotide sequence of contig_160 of TfOz-N36 SEQ ID NO: 341 Nucleotide sequence of contig_172 of TfOz-N36 SEQ ID NO: 342 Nucleotide sequence of contig_173 of TfOz-N36 SEQ ID NO: 343 Nucleotide sequence of contig_178 of TfOz-N36 SEQ ID NO: 344 Nucleotide sequence of contig_188 of TfOz-N36 SEQ ID NO: 345 Nucleotide sequence of contig_192 of TfOz-N36 SEQ ID NO: 346 Nucleotide sequence of contig_195 of TfOz-N36 SEQ ID NO: 347 Nucleotide sequence of contig_197 of TfOz-N36 SEQ ID NO: 348 Nucleotide sequence of contig_198 of TfOz-N36 SEQ ID NO: 349 Nucleotide sequence of contig_20 of TfOz-N36 SEQ ID NO: 350 Nucleotide sequence of contig_200 of TfOz-N36 SEQ ID NO: 351 Nucleotide sequence of contig_202 of TfOz-N36 SEQ ID NO: 352 Nucleotide sequence of contig_204 of TfOz-N36 SEQ ID NO: 353 Nucleotide sequence of contig_205 of TfOz-N36 SEQ ID NO: 354 Nucleotide sequence of contig_206 of TfOz-N36 SEQ ID NO: 355 Nucleotide sequence of contig_211 of TfOz-N36 SEQ ID NO: 356 Nucleotide sequence of contig_213 of TfOz-N36 SEQ ID NO: 357 Nucleotide sequence of contig_216 of TfOz-N36 SEQ ID NO: 358 Nucleotide sequence of contig_217 of TfOz-N36 SEQ ID NO: 359 Nucleotide sequence of contig_222 of TfOz-N36 SEQ ID NO: 360 Nucleotide sequence of contig_224 of TfOz-N36 SEQ ID NO: 361 Nucleotide sequence of contig_229 of TfOz-N36 SEQ ID NO: 362 Nucleotide sequence of contig_232 of TfOz-N36 SEQ ID NO: 363 Nucleotide sequence of contig_234 of TfOz-N36SEQ ID NO: 364 Nucleotide sequence of contig_235 of TfOz-N36 SEQ ID NO: 365 Nucleotide sequence of contig_242 of TfOz-N36 SEQ ID NO: 366 Nucleotide sequence of contig_244 of TfOz-N36 SEQ ID NO: 367 Nucleotide sequence of contig_247 of TfOz-N36 SEQ ID NO: 368 Nucleotide sequence of contig_249 of TfOz-N36 SEQ ID NO: 369 Nucleotide sequence of contig_256 of TfOz-N36 SEQ ID NO: 370 Nucleotide sequence of contig_258 of TfOz-N36 SEQ ID NO: 371 Nucleotide sequence of contig_260 of TfOz-N36 SEQ ID NO: 372 Nucleotide sequence of contig_261 of TfOz-N36 SEQ ID NO: 373 Nucleotide sequence of contig_264 of TfOz-N36 SEQ ID NO: 374 Nucleotide sequence of contig_265 of TfOz-N36 SEQ ID NO: 375 Nucleotide sequence of contig_268 of TfOz-N36 SEQ ID NO: 376 Nucleotide sequence of contig_271 of TfOz-N36 SEQ ID NO: 377 Nucleotide sequence of contig_272 of TfOz-N36 SEQ ID NO: 378 Nucleotide sequence of contig_273 of TfOz-N36 SEQ ID NO: 379 Nucleotide sequence of contig_275 of TfOz-N36 SEQ ID NO: 380 Nucleotide sequence of contig_278 of TfOz-N36 SEQ ID NO: 381 Nucleotide sequence of contig_280 of TfOz-N36 SEQ ID NO: 382 Nucleotide sequence of contig_290 of TfOz-N36 SEQ ID NO: 383 Nucleotide sequence of contig_297 of TfOz-N36 SEQ ID NO: 384 Nucleotide sequence of contig_30 of TfOz-N36 SEQ ID NO: 385 Nucleotide sequence of contig_301 of TfOz-N36 SEQ ID NO: 386 Nucleotide sequence of contig_302 of TfOz-N36 SEQ ID NO: 387 Nucleotide sequence of contig_304 of TfOz-N36 SEQ ID NO: 388 Nucleotide sequence of contig_31 of TfOz-N36 SEQ ID NO: 389 Nucleotide sequence of contig_315 of TfOz-N36 SEQ ID NO: 390 Nucleotide sequence of contig_319 of TfOz-N36 SEQ ID NO: 391 Nucleotide sequence of contig_321 of TfOz-N36 SEQ ID NO: 392 Nucleotide sequence of contig_327 of TfOz-N36 SEQ ID NO: 393 Nucleotide sequence of contig_330 of TfOz-N36 SEQ ID NO: 394 Nucleotide sequence of contig_334 of TfOz-N36 SEQ ID NO: 395 Nucleotide sequence of contig_335 of TfOz-N36 SEQ ID NO: 396 Nucleotide sequence of contig_336 of TfOz-N36 SEQ ID NO: 397 Nucleotide sequence of contig_338 of TfOz-N36 SEQ ID NO: 398 Nucleotide sequence of contig_347 of TfOz-N36 SEQ ID NO: 399 Nucleotide sequence of contig_353 of TfOz-N36 SEQ ID NO: 400 Nucleotide sequence of contig_360 of TfOz-N36 SEQ ID NO: 401 Nucleotide sequence of contig_375 of TfOz-N36 SEQ ID NO: 402 Nucleotide sequence of contig_378 of TfOz-N36 SEQ ID NO: 403 Nucleotide sequence of contig_385 of TfOz-N36 SEQ ID NO: 404 Nucleotide sequence of contig_389 of TfOz-N36 SEQ ID NO: 405 Nucleotide sequence of contig_392 of TfOz-N36 SEQ ID NO: 406 Nucleotide sequence of contig_400 of TfOz-N36 SEQ ID NO: 407 Nucleotide sequence of contig_402 of TfOz-N36 SEQ ID NO: 408 Nucleotide sequence of contig_406 of TfOz-N36 SEQ ID NO: 409 Nucleotide sequence of contig_409 of TfOz-N36 SEQ ID NO: 410 Nucleotide sequence of contig_410 of TfOz-N36SEQ ID NO: 411 Nucleotide sequence of contig_413 of TfOz-N36 SEQ ID NO: 412 Nucleotide sequence of contig_414 of TfOz-N36 SEQ ID NO: 413 Nucleotide sequence of contig_415 of TfOz-N36 SEQ ID NO: 414 Nucleotide sequence of contig_418 of TfOz-N36 SEQ ID NO: 415 Nucleotide sequence of contig_427 of TfOz-N36 SEQ ID NO: 416 Nucleotide sequence of contig_430 of TfOz-N36 SEQ ID NO: 417 Nucleotide sequence of contig_434 of TfOz-N36 SEQ ID NO: 418 Nucleotide sequence of contig_437 of TfOz-N36 SEQ ID NO: 419 Nucleotide sequence of contig_438 of TfOz-N36 SEQ ID NO: 420 Nucleotide sequence of contig_441 of TfOz-N36 SEQ ID NO: 421 Nucleotide sequence of contig_443 of TfOz-N36 SEQ ID NO: 422 Nucleotide sequence of contig_446 of TfOz-N36 SEQ ID NO: 423 Nucleotide sequence of contig_456 of TfOz-N36 SEQ ID NO: 424 Nucleotide sequence of contig_458 of TfOz-N36 SEQ ID NO: 425 Nucleotide sequence of contig_461 of TfOz-N36 SEQ ID NO: 426 Nucleotide sequence of contig_464 of TfOz-N36 SEQ ID NO: 427 Nucleotide sequence of contig_465 of TfOz-N36 SEQ ID NO: 428 Nucleotide sequence of contig_466 of TfOz-N36 SEQ ID NO: 429 Nucleotide sequence of contig_468 of TfOz-N36 SEQ ID NO: 430 Nucleotide sequence of contig_472 of TfOz-N36 SEQ ID NO: 431 Nucleotide sequence of contig_476 of TfOz-N36 SEQ ID NO: 432 Nucleotide sequence of contig_478 of TfOz-N36 SEQ ID NO: 433 Nucleotide sequence of contig_48 of TfOz-N36 SEQ ID NO: 434 Nucleotide sequence of contig_483 of TfOz-N36 SEQ ID NO: 435 Nucleotide sequence of contig_486 of TfOz-N36 SEQ ID NO: 436 Nucleotide sequence of contig_491 of TfOz-N36 SEQ ID NO: 437 Nucleotide sequence of contig_495 of TfOz-N36 SEQ ID NO: 438 Nucleotide sequence of contig_497 of TfOz-N36 SEQ ID NO: 439 Nucleotide sequence of contig_50 of TfOz-N36 SEQ ID NO: 440 Nucleotide sequence of contig_501 of TfOz-N36 SEQ ID NO: 441 Nucleotide sequence of contig_503 of TfOz-N36 SEQ ID NO: 442 Nucleotide sequence of contig_504 of TfOz-N36 SEQ ID NO: 443 Nucleotide sequence of contig_507 of TfOz-N36 SEQ ID NO: 444 Nucleotide sequence of contig_508 of TfOz-N36 SEQ ID NO: 445 Nucleotide sequence of contig_514 of TfOz-N36 SEQ ID NO: 446 Nucleotide sequence of contig_526 of TfOz-N36 SEQ ID NO: 447 Nucleotide sequence of contig_531 of TfOz-N36 SEQ ID NO: 448 Nucleotide sequence of contig_533 of TfOz-N36 SEQ ID NO: 449 Nucleotide sequence of contig_537 of TfOz-N36 SEQ ID NO: 450 Nucleotide sequence of contig_538 of TfOz-N36 SEQ ID NO: 451 Nucleotide sequence of contig_545 of TfOz-N36 SEQ ID NO: 452 Nucleotide sequence of contig_547 of TfOz-N36 SEQ ID NO: 453 Nucleotide sequence of contig_549 of TfOz-N36 SEQ ID NO: 454 Nucleotide sequence of contig_552 of TfOz-N36 SEQ ID NO: 455 Nucleotide sequence of contig_560 of TfOz-N36 SEQ ID NO: 456 Nucleotide sequence of contig_563 of TfOz-N36 SEQ ID NO: 457 Nucleotide sequence of contig_566 of TfOz-N36SEQ ID NO: 458 Nucleotide sequence of contig_57 of TfOz-N36 SEQ ID NO: 459 Nucleotide sequence of contig_574 of TfOz-N36 SEQ ID NO: 460 Nucleotide sequence of contig_583 of TfOz-N36 SEQ ID NO: 461 Nucleotide sequence of contig_588 of TfOz-N36 SEQ ID NO: 462 Nucleotide sequence of contig_594 of TfOz-N36 SEQ ID NO: 463 Nucleotide sequence of contig_596 of TfOz-N36 SEQ ID NO: 464 Nucleotide sequence of contig_599 of TfOz-N36 SEQ ID NO: 465 Nucleotide sequence of contig_600 of TfOz-N36 SEQ ID NO: 466 Nucleotide sequence of contig_607 of TfOz-N36 SEQ ID NO: 467 Nucleotide sequence of contig_610 of TfOz-N36 SEQ ID NO: 468 Nucleotide sequence of contig_614 of TfOz-N36 SEQ ID NO: 469 Nucleotide sequence of contig_623 of TfOz-N36 SEQ ID NO: 470 Nucleotide sequence of contig_627 of TfOz-N36 SEQ ID NO: 471 Nucleotide sequence of contig_633 of TfOz-N36 SEQ ID NO: 472 Nucleotide sequence of contig_634 of TfOz-N36 SEQ ID NO: 473 Nucleotide sequence of contig_636 of TfOz-N36 SEQ ID NO: 474 Nucleotide sequence of contig_641 of TfOz-N36 SEQ ID NO: 475 Nucleotide sequence of contig_655 of TfOz-N36 SEQ ID NO: 476 Nucleotide sequence of contig_659 of TfOz-N36 SEQ ID NO: 477 Nucleotide sequence of contig_661 of TfOz-N36 SEQ ID NO: 478 Nucleotide sequence of contig_662 of TfOz-N36 SEQ ID NO: 479 Nucleotide sequence of contig_672 of TfOz-N36 SEQ ID NO: 480 Nucleotide sequence of contig_678 of TfOz-N36 SEQ ID NO: 481 Nucleotide sequence of contig_683 of TfOz-N36 SEQ ID NO: 482 Nucleotide sequence of contig_685 of TfOz-N36 SEQ ID NO: 483 Nucleotide sequence of contig_686 of TfOz-N36 SEQ ID NO: 484 Nucleotide sequence of contig_690 of TfOz-N36 SEQ ID NO: 485 Nucleotide sequence of contig_695 of TfOz-N36 SEQ ID NO: 486 Nucleotide sequence of contig_701 of TfOz-N36 SEQ ID NO: 487 Nucleotide sequence of contig_702 of TfOz-N36 SEQ ID NO: 488 Nucleotide sequence of contig_703 of TfOz-N36 SEQ ID NO: 489 Nucleotide sequence of contig_708 of TfOz-N36 SEQ ID NO: 490 Nucleotide sequence of contig_71 of TfOz-N36 SEQ ID NO: 491 Nucleotide sequence of contig_710 of TfOz-N36 SEQ ID NO: 492 Nucleotide sequence of contig_713 of TfOz-N36 SEQ ID NO: 493 Nucleotide sequence of contig_717 of TfOz-N36 SEQ ID NO: 494 Nucleotide sequence of contig_720 of TfOz-N36 SEQ ID NO: 495 Nucleotide sequence of contig_725 of TfOz-N36 SEQ ID NO: 496 Nucleotide sequence of contig_726 of TfOz-N36 SEQ ID NO: 497 Nucleotide sequence of contig_729 of TfOz-N36 SEQ ID NO: 498 Nucleotide sequence of contig_730 of TfOz-N36 SEQ ID NO: 499 Nucleotide sequence of contig_732 of TfOz-N36 SEQ ID NO: 500 Nucleotide sequence of contig_737 of TfOz-N36 SEQ ID NO: 501 Nucleotide sequence of contig_739 of TfOz-N36 SEQ ID NO: 502 Nucleotide sequence of contig_745 of TfOz-N36 SEQ ID NO: 503 Nucleotide sequence of contig_748 of TfOz-N36 SEQ ID NO: 504 Nucleotide sequence of contig_749 of TfOz-N36SEQ ID NO: 505 Nucleotide sequence of contig_750 of TfOz-N36 SEQ ID NO: 506 Nucleotide sequence of contig_753 of TfOz-N36 SEQ ID NO: 507 Nucleotide sequence of contig_769 of TfOz-N36 SEQ ID NO: 508 Nucleotide sequence of contig_771 of TfOz-N36 SEQ ID NO: 509 Nucleotide sequence of contig_776 of TfOz-N36 SEQ ID NO: 510 Nucleotide sequence of contig_779 of TfOz-N36 SEQ ID NO: 511 Nucleotide sequence of contig_788 of TfOz-N36 SEQ ID NO: 512 Nucleotide sequence of contig_793 of TfOz-N36 SEQ ID NO: 513 Nucleotide sequence of contig_796 of TfOz-N36 SEQ ID NO: 514 Nucleotide sequence of contig_800 of TfOz-N36 SEQ ID NO: 515 Nucleotide sequence of contig_807 of TfOz-N36 SEQ ID NO: 516 Nucleotide sequence of contig_809 of TfOz-N36 SEQ ID NO: 517 Nucleotide sequence of contig_815 of TfOz-N36 SEQ ID NO: 518 Nucleotide sequence of contig_817 of TfOz-N36 SEQ ID NO: 519 Nucleotide sequence of contig_818 of TfOz-N36 SEQ ID NO: 520 Nucleotide sequence of contig_821 of TfOz-N36 SEQ ID NO: 521 Nucleotide sequence of contig_845 of TfOz-N36 SEQ ID NO: 522 Nucleotide sequence of contig_848 of TfOz-N36 SEQ ID NO: 523 Nucleotide sequence of contig_851 of TfOz-N36 SEQ ID NO: 524 Nucleotide sequence of contig_861 of TfOz-N36 SEQ ID NO: 525 Nucleotide sequence of contig_866 of TfOz-N36 SEQ ID NO: 526 Nucleotide sequence of contig_870 of TfOz-N36 SEQ ID NO: 527 Nucleotide sequence of contig_873 of TfOz-N36 SEQ ID NO: 528 Nucleotide sequence of contig_875 of TfOz-N36 SEQ ID NO: 529 Nucleotide sequence of contig_881 of TfOz-N36 SEQ ID NO: 530 Nucleotide sequence of contig_885 of TfOz-N36 SEQ ID NO: 531 Nucleotide sequence of contig_899 of TfOz-N36 SEQ ID NO: 532 Nucleotide sequence of contig_905 of TfOz-N36 SEQ ID NO: 533 Nucleotide sequence of contig_907 of TfOz-N36 SEQ ID NO: 534 Nucleotide sequence of contig_908 of TfOz-N36 SEQ ID NO: 535 Nucleotide sequence of contig_910 of TfOz-N36 SEQ ID NO: 536 Nucleotide sequence of contig_914 of TfOz-N36 SEQ ID NO: 537 Nucleotide sequence of contig_916 of TfOz-N36 SEQ ID NO: 538 Nucleotide sequence of contig_918 of TfOz-N36 SEQ ID NO: 539 Nucleotide sequence of contig_923 of TfOz-N36 SEQ ID NO: 540 Nucleotide sequence of contig_924 of TfOz-N36 SEQ ID NO: 541 Nucleotide sequence of contig_925 of TfOz-N36 SEQ ID NO: 542 Nucleotide sequence of contig_926 of TfOz-N36 SEQ ID NO: 543 Nucleotide sequence of contig_928 of TfOz-N36 SEQ ID NO: 544 Nucleotide sequence of contig_929 of TfOz-N36 SEQ ID NO: 545 Nucleotide sequence of contig_931 of TfOz-N36 SEQ ID NO: 546 Nucleotide sequence of contig_94 of TfOz-N36 SEQ ID NO: 547 Nucleotide sequence of contig_940 of TfOz-N36 SEQ ID NO: 548 Nucleotide sequence of contig_946 of TfOz-N36 SEQ ID NO: 549 Nucleotide sequence of contig_948 of TfOz-N36 SEQ ID NO: 550 Nucleotide sequence of contig_951 of TfOz-N36 SEQ ID NO: 551 Nucleotide sequence of contig_952 of TfOz-N36SEQ ID NO: 552 Nucleotide sequence of contig_957 of TfOz-N36 SEQ ID NO: 553 Nucleotide sequence of contig_959 of TfOz-N36 SEQ ID NO: 554 Nucleotide sequence of contig_96 of TfOz-N36 SEQ ID NO: 555 Nucleotide sequence of contig_966 of TfOz-N36 SEQ ID NO: 556 Nucleotide sequence of contig_969 of TfOz-N36 SEQ ID NO: 557 Nucleotide sequence of contig_978 of TfOz-N36 SEQ ID NO: 558 Nucleotide sequence of contig_983 of TfOz-N36 SEQ ID NO: 559 Nucleotide sequence of contig_984 of TfOz-N36 SEQ ID NO: 560 Nucleotide sequence of contig_988 of TfOz-N36 SEQ ID NO: 561 Nucleotide sequence of contig_990 of TfOz-N36 SEQ ID NO: 562 Nucleotide sequence of contig_991 of TfOz-N36 SEQ ID NO:563Nucleotide sequence of contig_1004 of TfOz5 SEQ ID NO:564Nucleotide sequence of contig_1008 of TfOz5 SEQ ID NO:565Nucleotide sequence of contig_1022 of TfOz5 SEQ ID NO:566Nucleotide sequence of contig_1028 of TfOz5 SEQ ID NO:567Nucleotide sequence of contig_1037 of TfOz5 SEQ ID NO:568Nucleotide sequence of contig_1041 of TfOz5 SEQ ID NO:569Nucleotide sequence of contig_1049 of TfOz5 SEQ ID NO:570Nucleotide sequence of contig_1053 of TfOz5 SEQ ID NO:571Nucleotide sequence of contig_1069 of TfOz5 SEQ ID NO:572Nucleotide sequence of contig_1071 of TfOz5 SEQ ID NO:573Nucleotide sequence of contig_1072 of TfOz5 SEQ ID NO:574Nucleotide sequence of contig_1074 of TfOz5 SEQ ID NO:575Nucleotide sequence of contig_1078 of TfOz5 SEQ ID NO:576Nucleotide sequence of contig_1086 of TfOz5 SEQ ID NO:577Nucleotide sequence of contig_1088 of TfOz5 SEQ ID NO:578Nucleotide sequence of contig_1106 of TfOz5 SEQ ID NO:579Nucleotide sequence of contig_112 of TfOz5 SEQ ID NO:580Nucleotide sequence of contig_1122 of TfOz5 SEQ ID NO:581Nucleotide sequence of contig_1124 of TfOz5 SEQ ID NO:582Nucleotide sequence of contig_1125 of TfOz5 SEQ ID NO:583Nucleotide sequence of contig_1139 of TfOz5 SEQ ID NO:584Nucleotide sequence of contig_1141 of TfOz5 SEQ ID NO:585Nucleotide sequence of contig_1158 of TfOz5 SEQ ID NO:586Nucleotide sequence of contig_1164 of TfOz5 SEQ ID NO:587Nucleotide sequence of contig_1165 of TfOz5 SEQ ID NO:588Nucleotide sequence of contig_1180 of TfOz5 SEQ ID NO:589Nucleotide sequence of contig_1184 of TfOz5 SEQ ID NO:590Nucleotide sequence of contig_1194 of TfOz5 SEQ ID NO:591Nucleotide sequence of contig_1206 of TfOz5 SEQ ID NO:592Nucleotide sequence of contig_1207 of TfOz5 SEQ ID NO:593Nucleotide sequence of contig_1215 of TfOz5 SEQ ID NO:594Nucleotide sequence of contig_1217 of TfOz5 SEQ ID NO:595Nucleotide sequence of contig_1225 of TfOz5 SEQ ID NO:596Nucleotide sequence of contig_1232 of TfOz5 SEQ ID NO:597Nucleotide sequence of contig_1244 of TfOz5 SEQ ID NO:598Nucleotide sequence of contig_1246 of TfOz5SEQ ID NO:599Nucleotide sequence of contig_1263 of TfOz5 SEQ ID NO:600Nucleotide sequence of contig_1266 of TfOz5 SEQ ID NO:601Nucleotide sequence of contig_1267 of TfOz5 SEQ ID NO:602Nucleotide sequence of contig_1270 of TfOz5 SEQ ID NO:603Nucleotide sequence of contig_1279 of TfOz5 SEQ ID NO:604Nucleotide sequence of contig_1281 of TfOz5 SEQ ID NO:605Nucleotide sequence of contig_129 of TfOz5 SEQ ID NO:606Nucleotide sequence of contig_1300 of TfOz5 SEQ ID NO:607Nucleotide sequence of contig_1319 of TfOz5 SEQ ID NO:608Nucleotide sequence of contig_1346 of TfOz5 SEQ ID NO:609Nucleotide sequence of contig_1350 of TfOz5 SEQ ID NO:610Nucleotide sequence of contig_1364 of TfOz5 SEQ ID NO:611Nucleotide sequence of contig_1366 of TfOz5 SEQ ID NO:612Nucleotide sequence of contig_138 of TfOz5 SEQ ID NO:613Nucleotide sequence of contig_141 of TfOz5 SEQ ID NO:614Nucleotide sequence of contig_143 of TfOz5 SEQ ID NO:615Nucleotide sequence of contig_153 of TfOz5 SEQ ID NO:616Nucleotide sequence of contig_17 of TfOz5 SEQ ID NO:617Nucleotide sequence of contig_173 of TfOz5 SEQ ID NO:618Nucleotide sequence of contig_176 of TfOz5 SEQ ID NO:619Nucleotide sequence of contig_178 of TfOz5 SEQ ID NO:620Nucleotide sequence of contig_179 of TfOz5 SEQ ID NO:621Nucleotide sequence of contig_183 of TfOz5 SEQ ID NO:622Nucleotide sequence of contig_187 of TfOz5 SEQ ID NO:623Nucleotide sequence of contig_194 of TfOz5 SEQ ID NO:624Nucleotide sequence of contig_199 of TfOz5 SEQ ID NO:625Nucleotide sequence of contig_2 of TfOz5 SEQ ID NO:626Nucleotide sequence of contig_204 of TfOz5 SEQ ID NO:627Nucleotide sequence of contig_210 of TfOz5 SEQ ID NO:628Nucleotide sequence of contig_214 of TfOz5 SEQ ID NO:629Nucleotide sequence of contig_217 of TfOz5 SEQ ID NO:630Nucleotide sequence of contig_220 of TfOz5 SEQ ID NO:631Nucleotide sequence of contig_224 of TfOz5 SEQ ID NO:632Nucleotide sequence of contig_237 of TfOz5 SEQ ID NO:633Nucleotide sequence of contig_245 of TfOz5 SEQ ID NO:634Nucleotide sequence of contig_246 of TfOz5 SEQ ID NO:635Nucleotide sequence of contig_252 of TfOz5 SEQ ID NO:636Nucleotide sequence of contig_262 of TfOz5 SEQ ID NO:637Nucleotide sequence of contig_266 of TfOz5 SEQ ID NO:638Nucleotide sequence of contig_268 of TfOz5 SEQ ID NO:639Nucleotide sequence of contig_270 of TfOz5 SEQ ID NO:640Nucleotide sequence of contig_276 of TfOz5 SEQ ID NO:641Nucleotide sequence of contig_281 of TfOz5 SEQ ID NO:642Nucleotide sequence of contig_285 of TfOz5 SEQ ID NO:643Nucleotide sequence of contig_286 of TfOz5 SEQ ID NO:644Nucleotide sequence of contig_288 of TfOz5 SEQ ID NO:645Nucleotide sequence of contig_291 of TfOz5SEQ ID NO:646Nucleotide sequence of contig_294 of TfOz5 SEQ ID NO:647Nucleotide sequence of contig_296 of TfOz5 SEQ ID NO:648Nucleotide sequence of contig_302 of TfOz5 SEQ ID NO:649Nucleotide sequence of contig_303 of TfOz5 SEQ ID NO:650Nucleotide sequence of contig_317 of TfOz5 SEQ ID NO:651Nucleotide sequence of contig_320 of TfOz5 SEQ ID NO:652Nucleotide sequence of contig_330 of TfOz5 SEQ ID NO:653Nucleotide sequence of contig_341 of TfOz5 SEQ ID NO:654Nucleotide sequence of contig_342 of TfOz5 SEQ ID NO:655Nucleotide sequence of contig_347 of TfOz5 SEQ ID NO:656Nucleotide sequence of contig_356 of TfOz5 SEQ ID NO:657Nucleotide sequence of contig_366 of TfOz5 SEQ ID NO:658Nucleotide sequence of contig_369 of TfOz5 SEQ ID NO:659Nucleotide sequence of contig_372 of TfOz5 SEQ ID NO:660Nucleotide sequence of contig_377 of TfOz5 SEQ ID NO:661Nucleotide sequence of contig_381 of TfOz5 SEQ ID NO:662Nucleotide sequence of contig_386 of TfOz5 SEQ ID NO:663Nucleotide sequence of contig_392 of TfOz5 SEQ ID NO:664Nucleotide sequence of contig_396 of TfOz5 SEQ ID NO:665Nucleotide sequence of contig_397 of TfOz5 SEQ ID NO:666Nucleotide sequence of contig_398 of TfOz5 SEQ ID NO:667Nucleotide sequence of contig_4 of TfOz5 SEQ ID NO:668Nucleotide sequence of contig_40 of TfOz5 SEQ ID NO:669Nucleotide sequence of contig_402 of TfOz5 SEQ ID NO:670Nucleotide sequence of contig_409 of TfOz5 SEQ ID NO:671Nucleotide sequence of contig_411 of TfOz5 SEQ ID NO:672Nucleotide sequence of contig_418 of TfOz5 SEQ ID NO:673Nucleotide sequence of contig_422 of TfOz5 SEQ ID NO:674Nucleotide sequence of contig_427 of TfOz5 SEQ ID NO:675Nucleotide sequence of contig_435 of TfOz5 SEQ ID NO:676Nucleotide sequence of contig_440 of TfOz5 SEQ ID NO:677Nucleotide sequence of contig_446 of TfOz5 SEQ ID NO:678Nucleotide sequence of contig_447 of TfOz5 SEQ ID NO:679Nucleotide sequence of contig_459 of TfOz5 SEQ ID NO:680Nucleotide sequence of contig_471 of TfOz5 SEQ ID NO:681Nucleotide sequence of contig_475 of TfOz5 SEQ ID NO:682Nucleotide sequence of contig_479 of TfOz5 SEQ ID NO:683Nucleotide sequence of contig_482 of TfOz5 SEQ ID NO:684Nucleotide sequence of contig_490 of TfOz5 SEQ ID NO:685Nucleotide sequence of contig_497 of TfOz5 SEQ ID NO:686Nucleotide sequence of contig_499 of TfOz5 SEQ ID NO:687Nucleotide sequence of contig_5 of TfOz5 SEQ ID NO:688Nucleotide sequence of contig_502 of TfOz5 SEQ ID NO:689Nucleotide sequence of contig_521 of TfOz5 SEQ ID NO:690Nucleotide sequence of contig_527 of TfOz5 SEQ ID NO:691Nucleotide sequence of contig_530 of TfOz5 SEQ ID NO:692Nucleotide sequence of contig_552 of TfOz5SEQ ID NO:693Nucleotide sequence of contig_559 of TfOz5 SEQ ID NO:694Nucleotide sequence of contig_56 of TfOz5 SEQ ID NO:695Nucleotide sequence of contig_561 of TfOz5 SEQ ID NO:696Nucleotide sequence of contig_565 of TfOz5 SEQ ID NO:697Nucleotide sequence of contig_572 of TfOz5 SEQ ID NO:698Nucleotide sequence of contig_574 of TfOz5 SEQ ID NO:699Nucleotide sequence of contig_577 of TfOz5 SEQ ID NO:700Nucleotide sequence of contig_580 of TfOz5 SEQ ID NO:701Nucleotide sequence of contig_584 of TfOz5 SEQ ID NO:702Nucleotide sequence of contig_594 of TfOz5 SEQ ID NO:703Nucleotide sequence of contig_616 of TfOz5 SEQ ID NO:704Nucleotide sequence of contig_62 of TfOz5 SEQ ID NO:705Nucleotide sequence of contig_625 of TfOz5 SEQ ID NO:706Nucleotide sequence of contig_631 of TfOz5 SEQ ID NO:707Nucleotide sequence of contig_637 of TfOz5 SEQ ID NO:708Nucleotide sequence of contig_646 of TfOz5 SEQ ID NO:709Nucleotide sequence of contig_65 of TfOz5 SEQ ID NO:710Nucleotide sequence of contig_655 of TfOz5 SEQ ID NO:711Nucleotide sequence of contig_659 of TfOz5 SEQ ID NO:712Nucleotide sequence of contig_662 of TfOz5 SEQ ID NO:713Nucleotide sequence of contig_671 of TfOz5 SEQ ID NO:714Nucleotide sequence of contig_672 of TfOz5 SEQ ID NO:715Nucleotide sequence of contig_691 of TfOz5 SEQ ID NO:716Nucleotide sequence of contig_701 of TfOz5 SEQ ID NO:717Nucleotide sequence of contig_709 of TfOz5 SEQ ID NO:718Nucleotide sequence of contig_719 of TfOz5 SEQ ID NO:719Nucleotide sequence of contig_721 of TfOz5 SEQ ID NO:720Nucleotide sequence of contig_723 of TfOz5 SEQ ID NO:721Nucleotide sequence of contig_725 of TfOz5 SEQ ID NO:722Nucleotide sequence of contig_736 of TfOz5 SEQ ID NO:723Nucleotide sequence of contig_737 of TfOz5 SEQ ID NO:724Nucleotide sequence of contig_765 of TfOz5 SEQ ID NO:725Nucleotide sequence of contig_768 of TfOz5 SEQ ID NO:726Nucleotide sequence of contig_775 of TfOz5 SEQ ID NO:727Nucleotide sequence of contig_788 of TfOz5 SEQ ID NO:728Nucleotide sequence of contig_796 of TfOz5 SEQ ID NO:729Nucleotide sequence of contig_799 of TfOz5 SEQ ID NO:730Nucleotide sequence of contig_804 of TfOz5 SEQ ID NO:731Nucleotide sequence of contig_806 of TfOz5 SEQ ID NO:732Nucleotide sequence of contig_81 of TfOz5 SEQ ID NO:733Nucleotide sequence of contig_813 of TfOz5 SEQ ID NO:734Nucleotide sequence of contig_816 of TfOz5 SEQ ID NO:735Nucleotide sequence of contig_821 of TfOz5 SEQ ID NO:736Nucleotide sequence of contig_823 of TfOz5 SEQ ID NO:737Nucleotide sequence of contig_825 of TfOz5 SEQ ID NO:738Nucleotide sequence of contig_837 of TfOz5 SEQ ID NO:739Nucleotide sequence of contig_841 of TfOz5SEQ ID NO:740Nucleotide sequence of contig_859 of TfOz5 SEQ ID NO:741Nucleotide sequence of contig_863 of TfOz5 SEQ ID NO:742Nucleotide sequence of contig_874 of TfOz5 SEQ ID NO:743Nucleotide sequence of contig_899 of TfOz5 SEQ ID NO:744Nucleotide sequence of contig_915 of TfOz5 SEQ ID NO:745Nucleotide sequence of contig_924 of TfOz5 SEQ ID NO:746Nucleotide sequence of contig_926 of TfOz5 SEQ ID NO:747Nucleotide sequence of contig_932 of TfOz5 SEQ ID NO:748Nucleotide sequence of contig_938 of TfOz5 SEQ ID NO:749Nucleotide sequence of contig_944 of TfOz5 SEQ ID NO:750Nucleotide sequence of contig_963 of TfOz5 SEQ ID NO:751Nucleotide sequence of contig_966 of TfOz5 SEQ ID NO:752Nucleotide sequence of contig_970 of TfOz5 SEQ ID NO:753Nucleotide sequence of contig_984 of TfOz5 SEQ ID NO:754Nucleotide sequence of contig_987 of TfOz5 SEQ ID NO:755Nucleotide sequence of contig_988 of TfOz5 SEQ ID NO:756Nucleotide sequence of contig_998 of TfOz5 Detailed description General Techniques and Definitions

[0050] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0051] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series.

[0052] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps,features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0053] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

[0054] Each feature of any particular aspect or example or example of the present disclosure may be applied mutatis mutandis to any other aspect or example or example of the present disclosure.

[0055] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0056] As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.

[0057] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0058] Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0059] By “consisting essentially of”, in the context of an amino acid sequence, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C-terminus. Moreover, in the context of a nucleotide sequence, the term “consisting essentially of”, is meant the recited nucleotide sequence together with an additional one, two or three nucleic acid residues or bases at the 5’ end or 3’ end thereof.

[0060] As used herein, the terms “approximately” and “about” refer to tolerances or variances associated with numerical values recited herein (e.g., ± 0.1%, 0.5%, 1.0%, 5.0% or 10%). The extent of such tolerances and variances are well understood by persons skilled in the art. Typically, such tolerances and variances do not compromise the structure, function and / or implementation of the compositions and methods described herein.

[0061] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[0062] For the present disclosure, the database accession number or unique identifier providedherein for a gene or protein, as well as the gene and / or protein sequence or sequences associated therewith, are incorporated by reference herein.

[0063] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims. Protozoal strains

[0064] The present disclosure is based on the identification of conserved protozoal strains of the species Tritrichomonas foetus. The inventors were able to isolate and sequence these novel protozoal strains of the species Tritrichomonas foetus from Australian cattle herds. Each strain was deposited on 27 November 2023 with the American Type Culture Collection (ATCC) located at ATCC Patent Depository, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA under the Budapest Treaty. The deposits, their taxonomic descriptions, dates of deposit and accession numbers have been provided as follows: Proposed Name Designation Date Deposited ATCC Patent Taxonomic Deposit Number Designation Tritrichomonas TfOz5 27 November 2023 PTA-127585 foetus (protozoa) Tritrichomonas TfOz36 (also 27 November 2023 PTA-127586 foetus (protozoa) referred to herein as TfOz-N36)

[0065] Accordingly, in a broad form, the present disclosure provides an isolated protozoal strain of the species Tritrichomonas foetus strain Oz5 (also referred to herein as TfOz5) having or deposited under ATCC Accession Number PTA-127585.

[0066] In another broad form, the present disclosure provides an isolated protozoal strain of the species Tritrichomonas foetus strain Oz-N36 (also referred to herein as TfOz-N36 or TfOz36) having or deposited under ATCC Accession Number PTA-127586.

[0067] For the purposes of this disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state (e.g., purified), or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0068] The term “genomic sequence” as used herein refers to a nucleotide sequence present in agenome. It is further envisaged that the term “genomic sequence” as used herein should be understood in a broad sense, that is, it may be a whole genome sequence or a part (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) of a whole genome sequence, such as a genomic sequence fragment. In particular examples, the genomic sequence is or comprises a chromosomal sequence. The term “chromosomal sequence” when used herein may refer to a nucleotide sequence present in a chromosome or chromosomal nucleic acid present in a cell. Again, it is contemplated that the term can refer a whole chromosomal sequence or a part of a chromosome sequence.

[0069] As used herein, a protozoal strain “derivative” refers to a strain derived from a reference strain (e.g., TfOz5 or TfOz-N36), that is, a strain produced using the reference strain as a starting point. Such derivatives may be modified, for example at the genetic level, without ablating the immunogenic activity or potential thereof. For example, clonally isolated, genetically engineered or otherwise mutated or genetically modified strains are an example of such derivatives. A derivative protozoal strain will suitably have comparable immunogenic activity to the Tritrichomonas foetus having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof (in particular TfOz5), or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof (in particular TfOz- N36), or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof (in particular TfOz5). In particular, a derivative protozoal strain will suitably elicit a comparable immune response, such as a protective immune response, to the Tritrichomonas foetus strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof. Moreover, a derivative protozoal strain can be utilised in methods of treating, preventing or ameliorating an infection with the Tritrichomonas foetus strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof. Further, a derivative protozoal strain will suitably have comparable immunogenic activity to the Tritrichomonas foetus strain deposited under ATCC Accession Number PTA-127585 or ATCC Accession Number PTA-127586.

[0070] Accordingly, in yet another broad form the present disclosure provides an isolated protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof.Reference to the nucleotide sequences set forth in SEQ ID NOs: 1 to 194 or a variant thereof suitably refers to the Tritrichomonas foetus strain TfOz5 or a derivative thereof.

[0071] In one broad form, the present disclosure provides an isolated protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 195 to 756, or a variant thereof. Reference to the nucleotide sequences set forth in SEQ ID NOs: 195 to 756 or a variant thereof suitably refers to the Tritrichomonas foetus strain TfOz-N36 or a derivative thereof.

[0072] In a further broad form, the present disclosure provides an isolated protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof. Reference to the nucleotide sequences set forth in SEQ ID NOs: 195 to 562 or a variant thereof suitably refers to the Tritrichomonas foetus strain TfOz-N36 or a derivative thereof.

[0073] In a further broad form, the present disclosure provides an isolated protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof. Reference to the nucleotide sequences set forth in SEQ ID NOs: 563 to 756 or a variant thereof suitably refers to the Tritrichomonas foetus strain TfOz-N36 or a derivative thereof.

[0074] Suitably, the nucleotide sequences of the isolated protozoal strains of the species Tritrichomonas foetus provided herein are at least partly obtained using long read genome sequencing, such as Oxford Nanopore Technologies (ONT) long read genome sequencing.

[0075] The polynucleotide sequences of SEQ ID NOs: 1 to 756 may be referred to herein as contigs. The term “contigs” as used herein, refers to contiguous regions of DNA sequence. “Contigs” can be determined by any number methods known in the art, such as, by comparing sequencing reads for overlapping sequences, and / or by comparing sequencing reads against databases of known sequences in order to identify which sequencing reads have a high probability of being contiguous. Contigs are often assembled from individual sequence reads or previously assembled sequence information in combination with sequence reads having overlapping end or edge sequence. Generally, but not exclusively, contigs comprise overlapping sequence reads that assemble into a larger sequence grouping, in many cases without intervening gaps or regions of undetermined sequence, or alternately without regions of known sequence and unknown length.

[0076] The term “assembly” of nucleic acid sequence data as used herein refers to the arrangement of singularly or independently provided sequence data, such as contigs, into a contiguous nucleotide sequence segment, such as a genomic sequence or chromosomal sequence. Any suitable procedure is contemplated for assembling the polynucleotide sequences or contigs described herein (e.g., SEQ ID NOs: 1 to 194 or 195 to 562 or 563 to 756) into a genomic sequence. Forexample, standard de novo assembly methods to reconstruct genomic or chromosomal sequences from component contigs. Such de novo assembly methods include, for example, the IDBA-UD algorithm of “IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth”, Peng Y et al., Bioinformatics.2012 Jun 1 ;28(11); the SPAdes method of “SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing”, Benkevich A et al., J Comput Biol. 2012 May; 19(5): 455-477; or the A5-miseq method of “A5-miseq: an updated pipeline to assemble microbial genomes from Illumina MiSeq data”, Coil D et al, Bioinformatics. 2015 Feb 15;31(4):587-9. In some examples, assembly includes positioning the polynucleotide sequences or contigs relative to each other by making reference to a reference genome. In particular examples, Pore-C sequencing is used to assemble the genomic sequence.

[0077] In some examples, the genomic sequence has been optimised by exclusion of repetitive sequences. In such examples, the genomic sequence is “masked”. As used herein, the term “masked” shall be understood to refer to the replacement of repetitive and low complexity nucleic acid regions with ‘N’s. As will be understood by the skilled person, a nucleotide denoted ‘N’ in a sequence may be any one of adenine, guanine, thymine, cytosine or no nucleotide. For example, SEQ ID NOs: 563 to 756 have been optimised by exclusion of repetitive sequences.

[0078] Protozoal strains of Tritrichomonas foetus that are closely related to the strain TfOz5 and / or TfOz-N36 would also be expected to be effective for eliciting an immune response, and treating, preventing or ameliorating a protozoal infection, such as a protozoal infection of the strain TfOz5 or TfOz-N36 described herein. Accordingly, the isolated protozoal strain of the present disclosure can include any variant or derivative thereof.

[0079] As used herein, a “variant” of a protozoal strain is one which shares a definable nucleotide sequence relationship with a reference nucleotide sequence (e.g., SEQ ID NOs: 1 to 756). The “variant” protozoal strain may have one or a plurality of nucleotides of the reference nucleotides deleted or substituted by different nucleotides. It is well understood in the art that some nucleotides may be substituted or deleted without changing the immunogenic potential of the protozoal strain (conservative substitutions). Suitably, protozoal strain variants share at least 70% or 75%, more particularly at least 80% or 85% or even more particularly at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 97.1%, 97.2%, 97.3%, 97.4% 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4% 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity with a reference nucleotide sequence, such as those nucleotide sequences set forth in SEQ ID NOs: 1 to 756, or a genomic sequence or a chromosomal sequence assembled therefrom.

[0080] In particular examples, the protozoal strain has a genomic sequence, such as a chromosomal sequence, that is at least 95%, 96%, 97%, 97.1%, 97.2%, 97.3%, 97.4% 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4% 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 1 to 194. In other examples, the protozoal strain has a genomic sequence, such as a chromosomal sequence, comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 1 to 194.

[0081] In particular examples, the protozoal strain has a genomic sequence, such as a chromosomal sequence, that is at least 95%, 96%, 97%, 97.1%, 97.2%, 97.3%, 97.4% 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4% 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 194 to 562. In some examples, the protozoal strain has a genomic sequence, such as a chromosomal sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 194 to 562.

[0082] In particular examples, the protozoal strain has a genomic sequence, such as a chromosomal sequence, that is at least 95%, 96%, 97%, 97.1%, 97.2%, 97.3%, 97.4% 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4% 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 563 to 756. In some examples, the protozoal strain has a genomic sequence, such as a chromosomal sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 563 to 756.

[0083] Terms used generally herein to describe sequence relationships between respective nucleotides include “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleotides may each comprise (i) only one or more portions of a complete nucleotide sequence that are shared by the nucleotides, and (ii) one or more portions which are divergent between the nucleotides, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 253389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0084] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA). In other examples, the sequence identity may be determined by Clustal Omega (https: / / www.ebi.ac.uk / jdispatcher / msa / clustalo).

[0085] Reference to cells or a population of cells of a protozoal strain of the species Tritrichomonas foetus as used herein encompasses any cells that have the same immunogenic and therapeutic efficacy characteristics as a protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof; or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof (in particular TfOz5 or TfOz-N36), and such cells and populations of cells are encompassed by the present disclosure. Such cells and populations of cells may also be inactivated or attenuated as described herein. Immunogenic compositions

[0086] The inventors have surprisingly shown for the first time that an immunogenic composition comprising an isolated protozoal strain of the species Tritrichomonas foetus, wherein the protozoal strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof; or nucleotide sequences set forth in SEQ ID NOs:195 to 562, or a variant thereof; or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof; or a Tritrichomonas foetus strain Oz5 deposited under ATCC Accession Number PTA-127585; or a Tritrichomonas foetus strain Oz-N36 deposited under ATCC Accession Number PTA-127586, shows an immunogenic effect in mammals. For example, the inventors have shown that an immunogenic composition or vaccine including the TfOz5 strain is capable of eliciting a protective immune response in cattle, and particularly in bulls. These findings have been exemplified in a pilot trial in infected bulls and non-infected bulls subsequently challenged with Tritrichomonas foetus. One benefit is that the identification and use of conserved protozoal strains of the species Tritrichomonas foetus provides efficacy in bulls, which primarily serve as reservoirs of Tritrichomonas foetus and thereby inhibit or prevent subsequent spread of this protozoal infection amongst breeding cows.

[0087] Accordingly, in a broad form the present disclosure provides an immunogenic composition comprising a protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof.

[0088] In a related form, the present disclosure provides an immunogenic composition comprising a protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof.

[0089] In a related form, the present disclosure provides an immunogenic composition comprising a protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

[0090] In another related form, the present disclosure provides an immunogenic composition comprising a Tritrichomonas foetus strain Oz5 deposited under the Budapest treaty and having ATCC Accession Number PTA-127585.

[0091] In yet a further related form, the present disclosure provides an immunogenic composition comprising a Tritrichomonas foetus strain Oz-N36 deposited under the Budapest treaty and having ATCC Accession Number PTA-127586.

[0092] As used herein, the term “immunogenic” will be understood to mean that the composition induces, elicits or generates an immune response. Suitably, the immune response is a protective immune response. By “protective immune response” is meant an immune response that is sufficient to prevent or at least reduce the severity or symptoms of an infection with a protozoal strain, such as Tritrichomonas foetus. A protective immune response may also be capable of at least partly treating, clearing or ameliorating an existing protozoal infection, such as in bulls or female cattle. As used herein, “elicits an immune response” or “induces an immune response” indicates the ability or potential of the immunogenic composition to elicit or generate an immuneresponse to the protozoal strain, upon administration to the mammal. As used herein, “immunize” and “immunization” refer to administering the immunogenic composition to elicit or potentiate a protective immune response to the protozoal strain.

[0093] In some examples, the protozoal strain of the species Tritrichomonas foetus is attenuated, inactivated and / or killed.

[0094] In particular examples, the protozoal strain is inactivated. The term “inactivate” means the protozoal strain has been modified in a way or killed such that the strain is incapable of reproduction in vivo or in vitro. Various physical and chemical methods of inactivating protozoal strains are known in the art, such as being irradiated (e.g., treated with UV, X-ray, electron beam or gamma radiation), heat treated (e.g., pasteurisation), or chemically treated (e.g., solvent / detergent treatment or acid treatment).

[0095] In certain examples, the protozoal strain of the species Tritrichomonas foetus is attenuated. The term “attenuate” means to modify the protozoal strain in a way that it becomes less virulent or pathogenic than prior to treatment. More particularly, this means that the protozoal strain exhibits a substantially reduced ability to cause a clinical disease while still being able to replicate in the host. With respect to attenuated protozoal strains, these are suitably included intact in the immunogenic composition.

[0096] In certain examples, the protozoal strain of the species Tritrichomonas foetus is killed.

[0097] In particular examples, the immunogenic composition comprises a single strain of the the protozoal strain of the species Tritrichomonas foetus. By way of example, the immunogenic composition comprises a protozoal strain of the species Tritrichomonas foetus strain TfOz5 (e.g., having ATCC Accession Number PTA-127585). In other examples, the immunogenic composition comprises a second protozoal strain of the species Tritrichomonas foetus strain TfOz- N36 (e.g., having ATCC Accession Number PTA-127586). In alternative examples, the immunogenic composition comprises a single protozoal strain of the species Tritrichomonas foetus strain TfOz-N36.

[0098] In certain examples, the immunogenic composition comprises two or more protozoal strains (e.g., 2, 3, 4, 5 etc.) of the species Tritrichomonas foetus. In certain examples, the immunogenic composition comprises a first protozoal strain of the species Tritrichomonas foetus strain TfOz5 and a second protozoal strain of the species Tritrichomonas foetus strain TfOz-N36. In particular examples, the protozoal strain of the species Tritrichomonas foetus is or comprises Tritrichomonas foetus strain TfOz5. In certain examples, the protozoal strain of the species Tritrichomonas foetus is or comprises Tritrichomonas foetus strain TfOz-N36. It is also contemplated that the immunogenic composition may comprise one or more further Tritrichomonas foetus strains, as are known in the art.

[0099] In some examples, the immunogenic composition comprises at least one protozoal strain (e.g., 1, 2, 3, 4, 5 etc.) of the species Tritrichomonas foetus and at least one additional antigen. In certain examples, the at least one additional antigen is a bacteria. For example, Aceinetobacter calcoaceticus, Acetobacter paseruianus, Actinobacillus pleuropneumoniae, Aeromonas hydrophila, Alicyclobacillus acidocaldarius, Arhaeglobus fulgidus, Bacillus pumilus, Bacillus stearothermophillus, Bacillus subtilis, Bacillus thermocatenulatus, Bordetella bronchiseptica, Burkholderia cepacia, Burkholderia glumae, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter hyointestinalis, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila spp., Chromobacterium viscosum, Erysipelothrix rhusiopathieae, Listeria monocytogenes, Ehrlichia canis, Escherichia coli, Haemophilus influenzae, Haemophilus somnus, Helicobacter suis, Lawsonia intracellularis, Legionella pneumophilia, Moraxellsa sp., Mycobactrium bovis, Mycoplasma hyopneumoniae, Mycoplasma mycoides subsp. mycoides LC, Clostridium perfringens, Odoribacter denticanis, Pasteurella (Mannheimia) haemolytica, Pasteurella multocida, Photorhabdus luminescens, Porphyromonas gulae, Porphyromonas gingivalis, Porphyromonas salivosa, Propionibacterium acnes, Proteus vulgaris, Pseudomonas wisconsinensis, Pseudomonas aeruginosa, Pseudomonas fluorescens C9, Pseudomonas fluorescens SIKW1, Pseudomonas fragi, Pseudomonas luteola, Pseudomonas oleovorans, Pseudomonas sp B11-1, Alcaliges eutrophus, Psychrobacter immobilis, Rickettsia prowazekii, Rickettsia rickettsia, Salmonella enterica all serovars, including for example: Salmonella enterica Typhimurium, Salmonella enterica Bongori, Salmonella enterica Dublin, Salmonella enterica Choleraseuis, and Salmonella enterica Newport, Serratia marcescens, Spirlina platensis, Staphlyoccocus aureus, Staphyloccoccus epidermidis, Staphylococcus hyicus, Streptomyces albus, Streptomyces cinnamoneus, Streptococcus uberis, Streptococcus suis, Streptomyces exfoliates, Streptomyces scabies, Sulfolobus acidocaldarius, Syechocystis sp., Vibrio cholerae, Borrelia burgdorferi, Treponema denticola, Treponema minutum, Treponema phagedenis, Treponema refringens, Treponema vincentii, Treponema palladium, Trueperella pyogenes and Leptospira species, such as the known pathogens Leptospira canicola, Leptospira grippotyposa, Leptospira hardjo, Leptospira borgpetersenii hardjo-bovis, Leptospira borgpetersenii hardjo-prajitno, Leptospira interrogans, Leptospira icterohaemorrhagiae, Leptospira pomona, and Leptospira bratislava, and combinations thereof.

[0100] In certain examples, the at least one additional antigen is a virus. For example, Avian herpesviruses, Bovine herpesviruses, Canine herpesviruses, Equine herpesviruses, Feline viral rhinotracheitis virus, Marek's disease virus, Ovine herpesviruses, Porcine herpesviruses, Porcine Epidemic Diarrhea virus (PEDv), Pseudorabies virus, Avian paramyxoviruses, Bovine respiratory syncytial virus, Canine distemper virus, Canine parainfluenza virus, canine adenovirus, canineparvovirus, Bovine Parainfluenza virus 3, Ovine parainfluenza 3, Rinderpest virus, Border disease virus, Bovine viral diarrhea virus (BVDV), BVDV Type I, BVDV Type II, Classical swine fever virus, Avian Leukosis virus, Bovine immunodeficiency virus, Bovine leukemia virus, Bovine tuberculosis, Equine infectious anemia virus, Feline immunodeficiency virus, Feline leukemia virus (FeLV), Newcastle Disease virus, Ovine progressive pneumonia virus, Ovine pulmonary adenocarcinoma virus, Canine coronavirus (CCV), pantropic CCV, Canine respiratory coronavirus, Bovine coronavirus, Feline Calicivirus, Feline enteric coronavirus, Feline infectious peritonitis, virus, Porcine epidemic diarrhea virus, Porcine hemagglutinating encephalomyletitis virus, Porcine parvovirus, Porcine Circovirus (PCV) Type I, PCV Type II, Porcine Reproductive and Respiratory Syndrome (PRRS) Virus, Transmissible gastroenteritis virus, Turkey coronavirus, Bovine ephemeral fever virus, Rabies, Rotovirus, Vesicular stomatitis virus, lentivirus, Avian influenza, Rhinoviruses, Equine influenza virus, Swine influenza virus, Canine influenza virus, Feline influenza virus, Human influenza virus, Eastern Equine encephalitis virus (EEE), Venezuelan equine encephalitis virus, West Nile virus, Western equine encephalitis virus, human immunodeficiency virus, human papilloma virus, varicella zoster virus, hepatitis B virus, rhinovirus, and measles virus, and combinations thereof.

[0101] In certain examples, the at least one additional antigen is a peptide antigen. For example, Bordetella bronchiseptica p68, gonadotropin-releasing hormone (GnRH), IgE peptides, Fel d 1, and cancer antigens, and combinations thereof. Examples of other antigens include nucleotides, carbohydrates, lipids, glycolipids, peptides, fatty acids, lipoteichoic and teichoic acid, and peptidoglycans, and combinations thereof.

[0102] In certain examples, the at least one additional antigen is a parasite. For example, Anaplasma, Fasciola hepatica (liver fluke), Coccidia, Eimeria spp., Neospora caninum, Toxoplasma gondii, Giardia, Dirofilaria (heartworms), Ancylostoma (hookworms), Cooperia, Haemonchus contortus (Barber pole worm) Ostertagia ostertagi (stomach worm), Dictyocaulus viviparous (lung worms), Trypanosoma spp., Leishmania spp., Trichomonas spp., Cryptosporidium parvum, Babesia, Schistosoma, Taenia, Strongyloides, Ascaris, Trichinella, Sarcocystis, Hammondia, Ixodes, Rhipicephalus, Dermacentor, Amblyomma, Boophilus, Hyalomma, Haemaphysalis species, and Isopsora, and combinations thereof.

[0103] It is envisaged that the immunogenic composition is formulated in dosage unit form to facilitate administration and ensure a uniform dosage thereof. Suitably, the immunogenic composition comprises at least about 5 x 101, 5 x 102, 5 x 103, 5 x 104, 5 x 105, 5 x 106, 5 x 107, 5 x 108, 5 x 109, 5 x 1010, 5 x 1011, 5 x 1012, 5 x 1013, 5 x 1014, 5 x 1015cells or cell equivalents (e.g., killed or lysed cells), or any range therein, of the protozoal strain per unit dose. In certain examples, the immunogenic composition comprises about 5 x 104to about 5 x 109cells or cell equivalentsof the protozoal strain per unit dose. More particularly, the immunogenic composition may comprise about 5 x 106to about 5 x 108cells or cell equivalents of the protozoal strain per unit dose. In various examples, the immunogenic composition comprises about 5 x 107cells or cell equivalents of the protozoal strain per unit dose.

[0104] Suitably, the immunogenic composition described herein comprises an adjuvant or an immunostimulatory agent, as are known in the art. The term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen (e.g., the isolated protozoal strain). Adjuvants may act primarily as a delivery system, primarily as an immune modulator or have features of both. Suitable adjuvants include those suitable for use in mammals, including humans, cattle and dogs.

[0105] Examples of known suitable delivery-system type adjuvants that can be used in mammals include, but are not limited to, calcium phosphate; squalane and squalene (or other oils of plant or animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Montanide ISA-50, Montanide ISA 61 VG (Seppic SA), anhydro mannitol ether octodecenoate, carbopol, Amphigen®, Amphigen® Mark II (Hydronics, USA), Alhydrogel® (BSA2; Accurate Scientific, Westbury, NY), Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacterium- derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); Bordetella pertussis antigens; tetanus toxoid; diphtheria toxoid; surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dicoctadecyl-N', N'bis(2- hydroxyethyl- propanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; peptides such as muramyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions such as BayolF / Arlacel A and water, or an emulsion of vegetable oil, water and an emulsifier such as lecithin; alum; bovine cytokines; cholesterol; additional oil emulsions, including a water in oil emulsion, and a water in oil in water emulsion; and mineral gels such as aluminium phosphate, alum (e.g., aluminum phosphate, aluminum sulfate or aluminum hydroxide); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; immunostimulatory DNA such as CpG DNA; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes (e.g., see International Publication WO2017 / 070735); ISCOM® and ISCOMATRIX® adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as muramyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE- Dextran alone or with aluminium phosphate; carboxypolymethylene such as Carbopol EMA; acrylic copolymer emulsions such as Neocryl A640 (e.g. U.S. Pat. No. 5,047,238); water in oilemulsifions, such as Montanide ISA 720, Montanide ISA 61 VG; oil-in-water emulsions, such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)); poly(D,L-laetide-co-glycolide) (PLG) microparticles or nanoparticles (e.g., the Next Adjuvant or NexaVAC™ System from the NA Vaccine Institute); ballistic particles; ceramic particles; polymeric particles; poliovirus, vaccinia or animal poxvirus proteins; or mixtures thereof.

[0106] Suitably, the adjuvant is or comprises a water in oil emulsion comprising a mineral oil. In particular examples, the adjuvant is or comprises a Montanide adjuvant. More particularly, the adjuvant is or comprises Montanide ISA 61 VG. Even more particularly, the adjuvant is or comprises anhydro mannitol ether octodecenoate. In particular examples, the immunogenic composition comprises the Tritrichomonas foetus strain of TfOz5 (e.g., the Tritrichomonas foetus strain having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or deposited under ATCC Accession Number PTA- 127585) and Montanide ISA 61 VG (anhydro mannitol ether octodecenoate). In other examples, the immunogenic composition comprises the Tritrichomonas foetus strain of TfOz-N36 (e.g., the Tritrichomonas foetus strain having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or deposited under ATCC Accession Number PTA-127586) and Montanide ISA 61 VG (anhydro mannitol ether octodecenoate). In further examples, the immunogenic composition comprises the Tritrichomonas foetus strain of TfOz5 (e.g., the Tritrichomonas foetus strain having a genomic sequence comprising or assembled from nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof or deposited under ATCC Accession Number PTA-127585) and Montanide ISA 61 VG (anhydro mannitol ether octodecenoate).

[0107] Suitably, the adjuvant has regulatory approval for administration to a mammal, such as cattle. Those skilled in the art will appreciate that the type of adjuvant is not limiting as long as it does not interfere with Beef Quality Assurance™.

[0108] The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts. The concentration of adjuvants useful in the context of the present disclosure can readily be determined by the skilled artisan.

[0109] Suitably, the adjuvant is present in an amount of about 10% to about 90% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or any range therein) by weight of the immunogenic composition. According to some examples, the adjuvant is present in an amount of about 40% to about 80%, more particularly about 50% to about 70% or even more particularly about 60% by weight of the immunogenic composition.

[0110] In a particular form, the immunogenic compositions described herein comprise one or more pharmaceutically acceptable carriers, diluents or excipients.

[0111] By “acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluent and excipients well known in the art may be used. These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.

[0112] A useful reference describing acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.

[0113] Any suitable procedure is contemplated for producing immunogenic or vaccine compositions. Exemplary procedures include, for example, those described in New Generation Vaccines (1997, Levine et al., Marcel Dekker, Inc. New York, Basel, Hong Kong), which is incorporated herein by reference. Producing a protozoal strain

[0114] The present inventors identified a method of successfully culturing and producing purified strains of Tritrichomonas foetus. Methods of the disclosure are applicable to small-, medium- and large-scale production methods. The methods are particularly useful for their ability to be scaled up for manufacturing pharmaceutical products at commercial scale. Methods for the production of protozoal strains of the disclosure will be apparent to the skilled artisan and / or described, for example, in Plastridge & Williams , J. Am. Vet. Med. Assoc.102: 89-95 (1943); Sutherland et al. Aust. Vet. J.29:67-69 (1953) and Buller & Corney, ANZSDP (2013).

[0115] Accordingly, in a broad form the present disclosure provides a method of producing a protozoal strain, said method including the step of culturing a protozoal strain described herein in a cell culture medium.

[0116] As used herein, the term “cell culture fluid” or “cell culture medium” will be understood to encompass the fluid or medium in which cells are grown for the purpose of producing a protozoal strain. The fluid or medium does not comprise the cells (e.g., the cells may have been removed, e.g., by centrifugation and / or removal of supernatant).

[0117] As used herein, the term “cell culture” will be understood to refer to the collective of the cell culture fluid or medium and the cultured cells.

[0118] The term “purify” or “purifying” or “purification” shall be taken to mean the removal,whether completely or partially, of at least one impurity present in the cell culture fluid, which thereby improves the level of purity of the protozoal strain.

[0119] The term “impurity” or “impurities” shall be taken to include one or more components in the cell culture fluid other than the protozoal strain. For example, impurities may include bacteria and yeast.

[0120] Tritrichomonas foetus is suitably cultivated in a medium suitable for cultivation of protozoal strains and for successful production. The cells can be cultivated in an adherent environment, e.g., while attached to a surface, or in a suspension environment, e.g., suspended in the medium. The medium may moreover be supplemented with additives known in the field, such as antibiotics, serum (notably fetal calf serum, etc.) and / or agar added in suitable concentrations. The medium may be supplemented with GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), Cell Boost™ 5, and / or an anticlumping agent. The medium used may comprise serum or be serum-free. Culture media for protozoal strains are known and include, for example, Trichomonas medium (Oxoid, ThermoFisher, Australia).

[0121] Suitably, the cell culture medium is or comprises Tritrichomonas foetus medium (TFM), as outlined in Buller and Corney, 2013 (“Bovine Trichomoniasis”), which is incorporated by reference herein. For illustration purposes, the composition and preparation method for TFM are provided below. TFM composition

[0122] Neutralised liver digest (Oxoid, cat # LP0027) 12.5 g ; Tryptose (Oxoid, cat # LP0047) 5 g ; Bacto agar (Oxoid, cat # LP0011) 1.5 g ; Sterile inactivated bovine serum (Sigma, cat # B 9433) 500 mL ; Penicillin (Sigma, cat # P 3032) 1.04 g ; Streptomycin (Sigma, cat # S 9137) 0.562 g ; Amphotericin B 0.0012 g; Gentamicin 0.016 g; and Distilled water 500 mL. Preparation of stock solutions

[0123] 1. Basal medium: In a 1 L bottle add 12.5 g neutralised liver digest and 5.0 g tryptose to 500 mL double distilled water. Adjust pH to 7.4 using HCl or NaOH. Add 1.5 g Bacto agar and heat to dissolve before autoclaving at 121°C for 15 minutes.

[0124] 2. Antibiotic solution: Add 1.04 g penicillin, 0.562 g streptomycin, 0.0012 g amphotericin B and gentamicin 0.016 g and dissolve. Filter sterilise through a 0.2 μm cellulose acetate filter. Preparation of TFM

[0125] Aseptically combine basal medium, sterile inactivated bovine serum, and antibiotic solution according to the table below. Dispense into sterile bottles or containers as required. Volumes for preparation of TFM Component 100 mL 500 mL 1000 mL Basal medium 50 mL 250 mL 500 mL Sterile inactivated bovine serum 50 mL 250 mL 500 mLAntibiotic solution 1 mL 5 mL 10 mL

[0126] Nystatin (200 units) can be added to each 1 mL of the medium immediately prior to use. Media can be stored at -20°C.50% foetal calf serum heat-inactivated at 56ºC for 1 hour, or 50% adult bovine serum heated to 65°C for 30 minutes removes natural agglutinins to T. foetus. Adult bovine serum inactivated at 65°C for 30 minutes is superior to media containing serum inactivated at 56°C for 30 minutes.

[0127] In view of the above, the TFM suitably comprises a base medium, an inactivated bovine serum, one or more antimicrobial agents, a neutralised liver digest and tryptose. In particular examples, the TFM contains no agar or is substantially free of agar (e.g., contains minimal agar, such as less than 0.15%, 0.1%, 0.05% or 0.01% agar by weight of the TFM).

[0128] The cell culture medium may further comprise at least one antimicrobial agent selected from the group consisting of an antibacterial agent, an antifungal agent, an antimycotic agent and combinations thereof. Non-limiting examples of suitable antimicrobial agents include amphotericin B, penicillin, streptomycin, gentamicin, tetracycline, normacin and nystatin.

[0129] The cell culture medium may further comprise a sugar, such as glucose and / or maltose. Suitably, the cell culture medium may comprise about 10 g / L to about 25 g / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 g / L), or any range therein, of maltose. In some examples, the cell culture medium may comprise about 10 g / L to about 25 g / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 g / L), or any range therein, of glucose.

[0130] In particular examples, the culture medium comprises a sugar, such as maltose and / or glucose, at a concentration of between about 10 g / L to about 25 g / L (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 g / L or any range therein) and an inactivated bovine serum at a concentration of between about 10 wt% to about 50 wt% (e.g., about 10, 15, 20, 25, 30, 45, 50 wt% or any range therein).

[0131] According to various examples, the cell culture medium is a liquid cell culture medium. To this end, the liquid cell culture medium can be suitable for suspension culture of the Tritrichomonas foetus strains provided herein. For example, agar is substantially absent from the media or the media only contains minimal agar. In one example, an agar plug is placed on top of the liquid medium to maintain anaerobic growth conditions.

[0132] Suitably, the cell culture medium may comprise about 0.6 µg / mL to about 4.8 µg / mL, or any range therein, of amphotericin B. In some examples, the cell culture medium may comprise about 0.1 µg / mL to about 0.3 µg / mL, or any range therein, of amphotericin B. In some examples, the cell culture medium may comprise about 895 µg / mL to about 1913 µg / mL, or any range therein, of penicillin. In some examples, the cell culture medium may comprise about 20,500 µg / mL to about 21,500 µg / mL, or any range therein, of penicillin. In particular examples, the cellculture medium may comprise about 322 µg / mL to about 2002 µg / mL, or any range therein, of streptomycin. In particular examples, the cell culture medium may comprise about 11,000 µg / mL to about 12,000 µg / mL, or any range therein, of streptomycin. In some examples, the cell culture medium may comprise about 8 µg / mL to about 256 µg / mL, or any range therein, of gentamicin. In some examples, the cell culture medium may comprise about 300 µg / mL to about 350 µg / mL, or any range therein, of gentamicin. In certain examples, the cell culture medium may comprise about 2.5 µg / mL to about 12.5 µg / mL, or any range therein, of tetracycline. In one example, the cell culture medium may comprise about 100 µg / mL to about 400 µg / mL, or any range therein, of normacin. In some examples, the cell culture medium may comprise about 20 µg / mL to about 40 µg / mL, or any range therein, of nystatin. Notwithstanding the above, it is envisaged that the protozoal strains can be cultured in the presence of any antimicrobial agent to reduce impurities.

[0133] In one example, the cell culture is carried out in batch, fed batch, continuous, semi- continuous, or perfusion mode. It will be apparent to the skilled person that reference to a batch, fed-batch, continuous and / or perfusion mode for a particular phase of cell culture (i.e., protozoal production) does not mean that the entire culture phase is carried out in that mode. For example, it only means that a period of the cell culture phase (e.g., at least 1 day) is carried out in that mode. It will also be understood that the mode does not necessary commence on day 0 of the culture phase. For example, the culture may commenced on day 0 and perfusion mode only commenced on day 2 of the cell culture phase.

[0134] Suitably, the suspension cell culture is operated in batch mode. It will be apparent to the skilled person that “batch mode” refers to a process where cells are initially cultured in a medium and this medium is neither removed, replaced, nor supplemented, i.e., the cells are not “fed” with new medium, during or before the end of cultivation.

[0135] In certain examples, the suspension cell culture is operated in fed-batch mode. It will be apparent to the skilled person that “fed-batch mode” refers to a process where one or more nutrients are fed to the bioreactor during the cultivation period. In one example, the protozoal production phase is operated in fed-batch mode.

[0136] In particular examples, the suspension cell culture is operated in perfusion mode. It will apparent to the skilled person that “perfusion mode” involves the constant feeding of fresh media and removal of spent media while retaining high numbers of viable protozoa (i.e., continuous media exchange).

[0137] It will be apparent to the skilled person from the disclosure herein that the protozoal strain is added to the cell culture medium as a single bolus feed, as multiple feeds, or continuously over the duration of the culture.

[0138] In one example, the cells are cultured in fluidized bed bioreactors, hollow fiber bioreactors,roller bottles, shake flasks, or stirred tank bioreactors.

[0139] In one example, the volume of the cell culture can be for example, about 0.01 L to about 0.1 L, or about 0.1 L to about 1 L, or about 1 L to about 5 L. In another example, the volume of the cell culture can be about 5 L to about 10 L, about 10 L to about 50 L, about 50 L to about 100 L, about 100 L to about 200 L, about 200 L to about 500 L, about 500 L to about 1000 L, about 1000 L to about 2000 L, or about 2000 L to about 5000 L. In one example, the volume of the cell culture is between about 35 and about 150 L. In one example, the volume of the cell culture is about 35 to about 150 L. In one example, the volume of the cell culture is about 50 to about 70 L.

[0140] For some examples, the cell culture is operated at a temperature that permits protozoal growth and production. For example, the cell culture has a temperature conventionally used in the state of the art for cultivating cells and producing protozoa. In one example, the cell culture of the protozoal strains is performed at a temperature of between about 20°C to about 39°C (e.g., about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or any range therein). For example, the cell culture of the protozoal strains is performed at a temperature of 30 ± 0.5°C or at a temperature of 31 ± 0.5°C.

[0141] Suitably, culturing of the protozoal strains described herein is performed under substantially anaerobic culture conditions. Such anaerobic culture conditions may include culturing the protozoal strains provided herein in the presence of no oxygen or minimal oxygen e.g., less than 5% oxygen (e.g., less than 5%, 4%, 3%, 2%, 1% or 0.5% oxygen or any range therein), more particularly less than 2% oxygen or even more particularly less than 1% oxygen).

[0142] In some examples, the present method further includes the step of at least partially isolating one or more cells or a population of cells of the protozoal strain from the cell culture medium.

[0143] In some examples, the present method further includes the step of inactivating or attenuating the one or more cells of the protozoal strain as described herein.

[0144] In another broad form, the present disclosure provides an immunogenic composition produced by the method described herein. Accordingly, the present method may include the further step of formulating the isolated or purified cells of the purified strain as an immunogenic composition. Methods of eliciting an immune response

[0145] One unexpected advantage of the present disclosure is that the immunogenic composition comprising the isolated protozoal strain of the species Tritrichomonas foetus as described herein shows an immunogenic effect in mammals. For example, the inventors have shown that the immunogenic composition treats, prevents and / or ameliorates a protozoal infection in cattle, and in particular in infected and non-infected bulls. The skilled artisan will appreciate that prior art bovine trichomonosis vaccines can reduce the clearance time of T. foetus from the reproductivetract in heifers, but are unable to induce protection or clear ongoing infections in bulls.

[0146] Accordingly, in one broad form, the present disclosure provides a method of eliciting an immune response in a mammal, said method including the step of administering a therapeutically effective amount of the immunogenic composition described herein, such as a first dose comprising a therapeutically effective amount of the immunogenic composition described herein, to the mammal to thereby elicit an immune response.

[0147] In a related form, there is provided the use of an immunogenic composition described herein in the manufacture of a medicament for eliciting an immune response in a mammal.

[0148] In another related form, there is provided an immunogenic composition described herein for use in eliciting an immune response in a mammal.

[0149] In a further related form, there is provided a method of treating, preventing or ameliorating a protozoal infection, inclusive of diseases, disorders and conditions associated therewith, in a mammal, said method including the step of administering a therapeutically effective amount of the immunogenic composition described herein, such as a first dose comprising a therapeutically effective amount of the immunogenic composition described herein, to the mammal to thereby treat, prevent or ameliorate the protozoal infection.

[0150] In yet a further related form there is provided the use of an immunogenic composition described herein in the manufacture of a medicament for treating, preventing or ameliorating a protozoal infection in a mammal.

[0151] In a related form, there is provided an immunogenic composition described herein for use in treating, preventing or ameliorating a protozoal infection in a mammal.

[0152] As generally used herein the terms “immunize”, “vaccinate” and “vaccine” refer to methods and / or compositions that elicit a protective immune response against a pathogen, whereby subsequent infection by the pathogen is at least partly prevented or minimized.

[0153] By “elicit an immune response” is meant generate or stimulate the production or activity of one or more elements of the immune system inclusive of the cellular immune system, humoral immune system (i.e., antibodies) and / or the native immune system.

[0154] Suitably, the immune response described herein includes one or more elements of the immune system, such as T lymphocytes, B lymphocytes, antibodies, neutrophils, dendritic cells inclusive of plasmacytoid dendritic cells, cytokines and / or chemokines. Non-limiting examples of cytokines include pro-inflammatory cytokines such as TNF-α, IL-6, IL-2, IL-6, IL-8, IL-17A and IL-1 (e.g., IL-1β). A non-limiting example of a chemokine is the neutrophil chemo-attractant IL- 8. In certain examples, the immune response that is elicited by the immunogenic composition is protective. In some examples, the immune response in the mammal includes activated B cells and / or T cells.

[0155] The first dose suitably includes a dosage of cells of the protozoal strain as that previously described herein. In certain examples, the first dose comprises about 5 x 104to about 5 x 109cells, more particularly about 5 x 106to about 5 x 108cells or even more particularly about 5 x 107cells of the protozoal strain.

[0156] As used herein “mammal” refers to any mammal capable of infection by the protozoal strain, such as a Tritrichomonas foetus, inclusive of humans, bovines, dogs, cats, pigs, deer, horses, donkeys, sheep and goats.

[0157] In some examples, the mammal is a non-human mammal. In this regard, the immunogenic composition may be considered a veterinary composition for use in the treatment, amelioration and / or prevention of infection with the protozoal strain in a domesticated mammal.

[0158] In particular examples, the mammal is bovine. As used herein, “bovines” are members of the mammalian sub-family Bovinae and include cattle, buffalo, bison and yaks. Cattle include all breeds and sub-species of the genus Bos, including Bos indicus and Bos taurus and hybrids thereof and inclusive of adult animals (e.g., cows, bulls), immature animals (e.g., heifers) and calves. According to certain examples, the mammal is a bull. For other examples, the mammal is a female bovine, such as a cow or a heifer. While both the terms “cow” and “heifer” refer to female bovines, the term “heifer” refers herein to any young female cow that has not given birth to a calf, typically one that has been weaned and is under the age of 3 years. The term “cow” often refers to an older female animal that has previously given birth to a calf.

[0159] The term “protozoal infection” as used herein relates to any protozoa, and more particularly any strain of Tritrichomonas foetus (e.g., TfOz5 and TfOz-N36 described herein) as are known in the art, that is capable of causing a disease, disorder or condition in an animal, such as an avian or mammal.

[0160] Suitably, the immunogenic composition described herein may also be effective in treating, preventing or ameliorating a disease, disorder or condition associated with the protozoal infection. Diseases, disorders and conditions caused by Tritrichomonas foetus in cattle, and more particularly cows, include transient vaginitis, cervicitis, endometritis, early embryonic mortality, abortion, infertility, embryonic losses, vaginal discharges, placental oedema, mild lymphocytic and histiocytic chorionitis and focal necrosis of trophoblasts, although without limitation thereto.

[0161] As hereinbefore described, the disclosure provides immunogenic compositions and / or use thereof for treating, preventing or ameliorating a protozoal infection, or a disease, disorder or condition associated therewith, in a mammal or other animal.

[0162] As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a protozoal infection by the protozoan, after it has begun to develop. Treatment need not be absolute to bebeneficial to the mammal. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

[0163] As used herein, “preventing”, “prevent” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, a protozoan and / or before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to prevent infection and / or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a mammal. A “prophylactic” treatment is a treatment administered to a mammal who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

[0164] Suitably, the disease, disorder or condition may be the result of a protozoal infection caused by Tritrichomonas. In particular examples, the methods herein relate to preventing, treating or ameliorating a protozoal infection caused by Tritrichomonas foetus, or a disease, disorder or condition associated therewith. In certain examples, the methods herein relate to preventing, treating or ameliorating a protozoal infection caused by the TfOz5 strain described herein, or a disease, disorder or condition associated therewith. In other examples, the methods herein relate to preventing, treating or ameliorating a protozoal infection caused by the TfOz-N36 strain described herein, or a disease, disorder or condition associated therewith. Booster immunization

[0165] The data herein further demonstrates a beneficial role for a second dose (i.e., a booster dose) comprising a therapeutically effective amount of the immunogenic composition described herein that is administered at a period of time after the first dose.

[0166] Accordingly, the aforementioned methods may include the further step of administering a second dose of the immunogenic composition comprising a therapeutically effective amount of the immunogenic composition, after the first dose. Similarly, the aforementioned medicaments and formulations may be formulated to be administered as a first dose prior to a second dose of the immunogenic composition comprising a therapeutically effective amount of the immunogenic composition. Further, the aforementioned medicaments and formulations may be formulated to be administered as a second dose after administration of a first dose of the immunogenic composition described herein.

[0167] Thus, in another broad form, the present disclosure relates to a method of eliciting an immune response in a mammal, said method including the steps of: (i) administering to the mammal a first dose comprising a therapeutically effective amount of the immunogenic composition described herein, and(ii) administering to the mammal a second dose comprising a therapeutically effective amount of the immunogenic composition described herein, at least 24 days after administration of the first dose.

[0168] A “booster immunization” as used herein, refers to a vaccination method where a target antigen is delivered at least twice to the mammal: i) first by a first dose comprising a first immunogenic composition that includes the target antigen or epitope; and ii) followed by a second dose comprising the immunogenic composition that includes the target antigen or epitope. Such a booster immunization strategy may include any number (e.g., 1, 2, 3, 4, 5 etc.) of boosting doses of the immunogenic composition administered at particular intervals of time after the first dose (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 52 weeks apart or any range therein).

[0169] In certain examples, a second dose will be administered to the mammal several days to several weeks after the first dose. In some examples, the second dose may be administered as late as one year after the first dose. To ensure sustained high levels of protection against disease, it may be helpful to re-administer a further dose (e.g., a third, fourth, fifth, six, seventh, eighth, ninth dose) to the mammal on a periodic basis. This periodic basis may range from monthly, to every six months, to yearly, to multiple years.

[0170] Suitably, the second dose is administered to the mammal at least about 14 days (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days) or at least about 1 month (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or any range therein) from the time of administration of the first dose. A further dose can then be administered to a mammal at regular intervals, such as about every 6 to 24 months, or about every 9 to 18 months, or for instance annually. For example, the second dose is administered to the mammal (for instance as a booster) at two month, six month or 1 year intervals.

[0171] In particular examples, a third dose will be administered to the mammal several days to several weeks after the second dose. In some examples, the third dose may be administered as late as one year after the second dose.

[0172] Suitably, the third dose is administered to the mammal at least about 14 days (e.g., at least about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days or any range therein) or at least about 1 month (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months or any range therein) from the time of administration of the second dose.

[0173] In particular examples, a second dose of the immunogenic composition is administered to the subject about 1 month after administration of a first dose of the immunogenic composition and a third dose of the immunogenic composition is administered to the subject about 12 months afteradministration of the first dose of the immunogenic composition.

[0174] Suitably, any booster dose (e.g., second dose, third dose etc) includes a dosage of cells of the protozoal strain as that previously described herein. In certain examples, the booster dose comprises about 5 x 104to about 5 x 109cells, more particularly about 5 x 106to about 5 x 108cells or even more particularly about 5 x 107cells of the protozoal strain. It is contemplated that any booster dose can include a dosage of cells of the protozoal strain that is the same or different to that of the first dose or any other earlier dose thereof.

[0175] Suitably, administration of the second dose is timed to coincide with times of maximal challenge or infection with the protozoal strain and / or risk of abortion. In particular examples, the second dose is administered to the mammal (e.g., to heifers / cows and / or bulls) about 1 week (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 weeks or any range therein) prior to a breeding season.

[0176] Any safe route of administration may be employed, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intranasal, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal and transdermal administration, although without limitation thereto.

[0177] In particular examples, the immunogenic compositions described herein are administered parenterally, such as subcutaneously or intramuscularly. In some examples, the immunogenic compositions described herein are administered intrapreputially.

[0178] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and / or microspheres.

[0179] Compositions may be presented as discrete units such as capsules, sachets, functional foods / feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimatelyadmixing the agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0180] The above compositions may be administered in a manner compatible with the dosage formulation, and in such an amount as effective (e.g., capable of eliciting a protective immune response). The dose administered to a subject, in the context of the present disclosure, should be sufficient to effect a beneficial response in a subject over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

[0181] In a particular example, the composition is suitable for parenteral administration to a subject.

[0182] So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples. EXAMPLES Example 1. Methodology Culture collection

[0183] During 2020-2021, a collection of ~66 isolates from 20 different herds in Queensland and the Northern Territory identified as T. foetus qPCR positive were collected into Trichomonasmedium (Oxoid, ThermoFisher, Australia) and transported at 22-37°C to The Queensland Alliancefor Agriculture and Food Innovation (QAAFI) laboratories, The University of Queensland (UQ) St Lucia campus. These were incubated at 37°C for 4-5 days prior to mixing the culture pellets with T. foetus freezing medium [Foetal calf serum (10%), RPMI 1640 (with L glutamine) (80%) and Dimethylsulphoxide (DMSO) (10%)] and stored at -80°C as described by Buller & Corney (2013).

[0184] A laboratory strain (YVL-W) from Biosecurity Science Laboratories (BSL, QLD Department of Agriculture & Fisheries) was used to test culture and freeze-thawing conditions prior to growth of the frozen field isolates. YVL-W culture is routinely maintained by BSL at room temperature on the bench and sub-cultured once per week and it was provided to QAAFI with an unknown commensal bacterial contaminant.

[0185] Cultures were confirmed by microscopy (100x) and using qPCR of boiled lysates and or DNA extracts as described previously (McMillen & Lew 2006). Isolates selected as the vaccine and challenge strains in this study were: TfOz5 originated from southwest Queensland property (Morney Plains Station, intersection of Diamantina River and Farrars Creek), and TfOz-N36 originated from the Barkly Tablelands of the Northern Territory (Brunchilly Station, NT). Genomic sequencing

[0186] It was demonstrated previously that T. foetus of bovine and porcine origin cluster together (100%) compared to those of feline origin (99%, 1% difference with bovine / porcine isolates) when comparing the sequences of nine cysteine proteases, cytosolic malate dehydrogenase and the internal transcribed spacer region 2 as a control sequence (Slapeta et al.2012). Currently there is very little genomic data for T. foetus (estimated genome size 160Mb), with data available only from the T. foetus K strain, the T. foetus Belfast strain and the T. foetus KV-1 strain (ATCC 30924; GenBank Assembly No. GCA_041260205.1).

[0187] The T. foetus K strain (the ‘reference genome’) was sequenced using short read next generation sequencing (NGS) methods (Illumina and 454) with 3,730 contigs (1,480 scaffolds) and 67.6 Mbp of data (Benchimol et al. 2017); GenBank accession: GCA_001839685.1; 25 / 10 / 2016). The T. foetus Belfast strain was sequenced using PacBio long read NGS methodology resulting in 2,776 scaffolds and 147Mb of data (unpublished, GenBank accession: GCA_905133005.1; 25 / 01 / 2021).

[0188] Instead of analysing the ten loci which were shown to be 100% conserved for T. foetus strains of bovine origin, Oxford Nanopore Technologies (ONT) long read genome sequencing (in house) was used to analyse the two Australian isolates selected for the vaccination and challenge experiments. Methods for DNA extraction and ONT analysis were first established using the Biosecurity lab strain YVL-W.

[0189] Tritrichomonas foetus YVL-W cells (50 mL) were grown in agar-free TFM for 2 days at 30°C. The cultures were centrifuged at 1,000 x g for 10 mins at 4°C and washed in 50 mL ice-cold PBS twice. The cell pellets were resuspended in 1 mL PBS and centrifuged 1,000 x g for 10 mins at 4°C, the supernatant removed, and the dry pellets frozen at -20°C overnight. One millilitre lysis buffer (100 mM Tris pH9.0, 100 mM NaCl, 10 mM EDTA, 1.5 % SDS) was added to the pellet and thawed on ice until complete cell lysis was observed. One mL Phenol-Chloroform-Isoamyl alcohol (25:24:1) was added immediately and mixed by inversion. Incubate at 4°C for 20 min, with shaking at 900 rpm. The samples were centrifuged at 14,800 g for 10 mins at 4°C and the aqueous phase transferred into a new tube.

[0190] The DNA was precipitated with two volumes of ethanol and incubating at -20 °C for 30 min. The samples were centrifuged at 14,800 x g for 20 mins at 4°C; the supernatant was removed and 500 µL 70 % ethanol was added. The samples were centrifuged at 14,800 x g for 10 mins at 4°C; the supernatant removed, and the DNA pellet allowed to air dry for 5 min. The pellet was resuspended in 50 µL double distilled water (ddH2O) and incubated with 50 µg RNase overnight at room temperature. The DNA was precipitated with two volumes of ethanol and 0.1 volumes of 3M Na-Acetate (pH 5.2); incubating at -20°C for 30 min. The samples were centrifuged at 14,800 g for 20 mins at 4°C; the supernatant was removed and 500 µL 70 % ethanol was added. Thesamples were centrifuged at 14,800 g for 10 mins at 4°C; the supernatant removed, and the DNA pellet allowed to air dry for 5 min. The DNA was resuspended in 50 µL ddH2O and analysed by gel electrophoresis, spectrophotometry and Qubit (Bio-Rad). Purified DNA (5 µg) was used for long-read sequencing with the Nanopore Ligation sequencing gDNA kit (SQK-LSK 110), following the manufacturer’s instructions.

[0191] The above methods were repeated for T. foetus TfOz5 and TfOz-N36 isolates once cultures were established as described below. Following assembly and annotation, the ten sequences previously used to cluster T. foetus strains of different host origins were assembled and compared to confirm conservation of the Australian isolates to that previously published for YVL-W Biosecurity strain (Slapeta et al.2012).

[0192] The whole-genome average nucleotide identity (ANI) of the two T. foetus field isolate genomes (TfOz5 and TfOz-N36) was computed using FastANI 1.33. Prokka 1.14.6, a two-step wrapper that incorporated Prodigal 2.6.3, was utilised initially to annotate the genome sequences. As Prokka is primarily a prokaryotic genome annotation tool, base-calling was repeated with guppy version 6 using standard accuracy, but these assemblies contained many errors. Further refinement was done using the super high accuracy base-calling algorithm, but these contained several small contigs that contained 200-10,000 bp, which aligned back into larger contigs. These small contigs were removed by using NanoFilt to filter the fastq file, quality: Q12 and sequences > 20,000 bp; The Flye assembler was used to assemble the data, and, any contigs with less than 20,000 bp were removed. Optimisation of culture protocols and frozen storage of TfOz isolates

[0193] Initial methods used for culture optimisation using TFM (Plastridge & Williams 1943; Sutherland et al. 1953) were prepared as outlined in the Australian and New Zealand Standard Diagnostic Procedures (Buller & Corney 2013). The stored field isolates were heavily contaminated with bacteria and yeast and thus antimicrobial concentrations were increased and added to TFM to clear the contaminations to obtain clean pure seed stocks of T. foetus isolates TfOz5 and TfOz-N36.

[0194] Briefly, 12.5 g / L neutralised liver extract, 5 g / L tryptose and 1.5 g / L Bacto agar were dissolved in ddH2O, pH adjusted to 7.4 and autoclaved for 15 mins at 121°C. The media was allowed to cool, and 50% inactivated bovine serum was added to the media for complete TFM. Antibiotics and antimycotics were added as required depending on the contamination (see Table 1). Yeast and fungi were viewed using microscopy and the presence of bacteria was screened using blood agar plates. Table 1. Antimicrobials used to treat the field Tritrichomonas foetus isolate cultures.Antimicrobial Target Supplier Maintenance Range of concentration Concentrations used Amphotericin Antifungal Thermo- 1.2 µg / mL 0.6 - 4.8 µg / mL B Fisher Penicillin Antibacterial 1040 µg / mL 895 - 1913 µg / mL Streptomycin Antibacterial 562 µg / mL 322 - 2002 µg / mL Gentamicin Antibacterial Thermo- 16 µg / mL 8 - 256 µg / mL Fisher Tetracycline Antibacterial 2.5 µg / mL 2.5 – 12.5 µg / mL Normacin Broad range:Fungal, InvivoGen 200 µg / mL 100 – 400 Bacterial,Mycoplasma µg / mL Nystatin Antifungal Thermo- 20 µg / mL 20 – 40 µg / mL Fisher

[0195] Once pure cultures were established, field T. foetus isolates are maintained in TFM supplemented with 1040 µg / mL penicillin, 562 µg / mL streptomycin, 1.2 µg / mL amphotericin B and 16 µg / mL gentamicin which is higher than normal TFM medium which routinely uses 750 μg / mL of penicillin and 82 μg / mL streptomycin only (nystatin 200 units optional) (Buller & Corney 2013). When liquid cultures were required (for DNA extraction or for vaccine and challenge strain preparation), agar was omitted from the media due to problematic harvest of pure T. foetus cells in the presence of the agar, and an agar plug was layered on top of the liquid medium to provide the anaerobic conditions for growth without the agar interfering with harvesting. Small pilot vaccination and treatment trial

[0196] UQ Animal Ethics approval is under Project Number 2021 / AE000410 entitled ‘Res- Experimental efficacy and safety of a vaccine to prevent trichomoniasis in beef bulls’ with CI Dr Gry Brandt Boe-Hansen. Two trial experiments were undertaken, the first was a vaccination trial using T. foetus negative bulls and the second was a treatment trial using T. foetus positive bulls. The bulls were provided by Hancock Ag following qPCR pre-screening for T. foetus (McMillen & Lew 2006) and Campylobacter fetus subspecies venerealis (Tabor et al. 2021, MDC P.PSH.0799 Milestone 7 report, October 2021) prior to transport to UQ’s Pinjarra Hills farm in January 2022. After arrival, the bulls were re-sampled to confirm T. foetus qPCR results. Pilot vaccination challenge trial

[0197] T. foetus negative bulls were vaccinated (n=6) twice 4 weeks apart with killed cultured T. foetus (Strain TFOz5) cells and control cattle (n=6) received a mock adjuvant PBS mix. Cultured cells 1 x 109cells from 48 x 40 mL TfOz5 cultures were washed with sterile PBS to remove culture media with a final resuspension of 14 mL PBS. This mixture was heated to 65°Cfor 40 minutes to inactivate the cells. Cell death was examined visually under a microscope and confirmed by negative culture growth. The vaccine was prepared by homogenising 14 mL of washed inactivated cells with 26 mL of Montanide ISA 61 VG (Seppic Australia) adjuvant (60:40 w / w ratio of adjuvant to antigen). Vaccine doses (2 mL; 5 x 107cells per dose) were prepared in 5 mL syringes and stored at 4°C until required. Vaccines were delivered subcutaneously using 18G needles. This process was repeated for the preparation of the second dose.

[0198] Challenge was undertaken using a T. foetus isolate from a different herd to avoid potential homogeneous challenge. T. foetus (Strain TFOz-N36; 8 x 108cells) cells were washed with sterile PBS to remove culture media with a final resuspension of 60 mL PBS. The cells were examined for viability under a microscope. Two weeks following the second vaccine dose, bulls were challenged with 3 mL T. foetus (4.1 x 107live cells / dose), administered through an Artificial Insemination (AI) catheter into the prepuce. Presence of T. foetus was subsequently confirmed using direct T. foetus qPCR and culture fortnightly. A second challenge was applied to half of the bulls approximately five months after vaccination using 8 x 107live cells / dose, with qPCR and culture monitoring occurring monthly for another three months.

[0199] At the end of the trial, the serum IgG serum collected was screened using ELISA. Blood was collected from tail veins using clot activators / gel Vacutainers with 18 or 20 G needles. The blood was allowed to clot overnight, followed by centrifugation to separate the sera. Whole T.foetus cells (5 x 104 in 50 µL PBS) were added to each well of a Maxisorp plate and allowed tobind for 4 hours. The plates were washed twice with 200 µL 95% ethanol / well and allowed to air dry. PBS-T (Phosphate buffered saline with 0.05% Tween-20) with 5% inactivated horse serum was used to block plates (200 µL / well), overnight at 37 °C. Serum samples were diluted 1:1,000 in blocking solution; 100 µL was added to the wells and the plate incubated at 37 °C for 90 minutes. HRP bound secondary antibodies against bovine IgG (Anti-bovine IgG – peroxidase, Sigma #A5295) was used at 1:100,000 in blocking solution; 100 µL was added to the wells and the plate incubated at 37 °C for 30 minutes. Signal was detected using TMB substrate kit (ThermoFisher Cat# 34021), stopped with 1 N HCl and read at 450 nm using the Multiskan FC Microplate Photometer (Thermo Fisher). Pilot treatment trial

[0200] Ten T. foetus positive bulls were treated using the vaccine as described above using two doses of vaccine as above. Presence or absence of T. foetus was subsequently confirmed using direct T. foetus qPCR and culture followed by microscopy and qPCR. Bull sampling was approximately every two weeks. Example 2. Results Identifying a conserved Australian strain of Tritrichomonas foetus (TfOz)

[0201] Several isolates were stored and two were selected for vaccination and challenge from two different herds in Queensland (TfOz5) and the Northern Territory (TfOz-N36). Long read sequencing methods require intact long strands of DNA unlike short sequences for qPCR detection. Kits routinely used to generate long DNA strands from bacteria or mammals, gave very poor DNA yields for T. foetus extracts that were not usable for ONT sequencing. Attempts to isolate the nuclear DNA to avoid the cytosolic nuclease degradation and standard lysis and phenol chloroform methods were unsuccessful (Figure 1A).

[0202] Research literature confirms that trichomonads have high levels of lysosomal hydrolytic activities and strong endogenous nucleases which degrade DNA after cell lysis (Wang & Wang 1985; Honigberg & Mohn 1973; Riley & Krieger 1992). The methods applied by these papers included the use of centrifugation gradients and outdated reagents and no new publicly available DNA extraction methods for trichomonad genomics sequencing is published. As a result a new method was developed to isolate T. foetus high molecular weight DNA by using alkaline lysis at 4°C as these endogenous nucleases are less active under these conditions (Figure 1B).

[0203] The generated genomic sequence data was better than all previously attempted genome sequencing projects for T. foetus and T. vaginalis with 10-100 fold less number of contigs generated. Table 2 shows the outputs of available genome projects compared with our ONT genomic assembly statistics.

[0204] Conservation of T. foetus bovine isolates based on the 10 loci previously sequenced demonstrated conservation as predicted for bovine T. foetus isolates (Slapeta et al.2012), data not shown. Based on the Average Nucleotide Identity (ANI) estimate between the two T. foetus strains TfOz5 (vaccine strain) and TfOz-N36 (challenge strain) the genomic sequence similarity was estimated at 99.2%. Table 2. Improved Oxford Nanopore Technologies long read assembly statistics for Tritrichomonas foetus isolates YVL-W, TfOz5 and TfOz-N36 compared with available genomic sequences. T. foetus Sequencing No. of Total length GC (%) Reference Strain method Contigs (Mbp) YVL-W Oxford NanoPore 226* 111.7 30.63 This study TfOz5 Oxford NanoPore 194ƚ110 30.56 This study TfOz-N36 Oxford NanoPore 368ǂ104.7 30.63 This study ATCC30924 Oxford Nanopore; 693 148.7 31 Abdel-Glil et Strain KV-1 Illumina MiSeq; al.2024 HiC Reference Illumina HiSeq and 3730 67.6 31 Benchimol et Strain K 4544 GS-FLX al.2017 Belfast PacBio 2776 147 30.5 Unpublished, GenbankTrichomonas Shotgun 17,290 176.4 32.7 Carlton et al. vaginalis scaffolds 2007 Improved Nanopore assemblies: *decreased from 485 contigs; ƚ decreased from 248 contigs; ǂ decreased from 535 contigs Culture optimisation of culture protocols and frozen storage of TfOz isolates

[0205] The revived field isolates were contaminated with bacteria, yeast and fungi. TFM uses low concentrations of penicillin and streptomycin which may be acceptable for short-term diagnostic applications but not for long-term storage of cultures for vaccine development. As a result, high concentrations of several antimicrobials were added to develop T. foetus clean cultures for future seed development.

[0206] All the isolates grew differently in terms of growth rate as determined by microscopy. The lab strain YVL-W was able to tolerate some aerobic growth conditions whereas field isolates TfOz5 and TfOz-N36 could not. The methods developed here for growth and scale up required months of optimisation. When first introduced into culture, field isolates grew very slowly, were highly contaminated, and needed repeated treatments and passages to adapt to in vitro growth conditions.

[0207] The agar content in the media impeded DNA extraction and preparation of doses for vaccination and challenge, however using liquid TFM with an agar plug overcame this issue and the protozoa could be easily extracted from the liquid without interference from agar clumps. Small pilot vaccination and treatment trial

[0208] All bulls were C. fetus subsp. venerealis negative prior to delivery to Pinjarra Hills farm with a mixture of T. foetus qPCR negative and positive bulls. There was a slight delay to the administration of the second vaccine dose, as a result it was administered at 32 days instead of 28 days. Table 3 summarises the qPCR and culture results from the pilot vaccination trial. Of the six vaccinated bulls, three were positive at one week following challenge with one of these subsequently negative by two weeks post challenge. Three vaccinated bulls were consistently negative post challenge.

[0209] Conversely, for the un-vaccinated group, three were not immediately positive following challenge, with three positive post challenge. Results are presented in Table 3 show two vaccinated bulls as persistently negative, one persistently positive, and three as transiently negative with increasing positivity after the second challenge. Based on these four out of the six vaccinated bulls, the efficacy was calculated at 67% vaccination success rate in old bulls. For unvaccinated bulls, four out of the six were consistently positive either after the first and second challenge or after the second challenge. The other two were only transiently positive. IgG ELISA analysis demonstrated a consistent response to vaccination (Figure 2).Table 3. qPCR and culture summary sampling following vaccination (n=6 vaccinated with two doses of killed T. foetus TfOz5) and two live challenges (live T. foetus TfOz-N36) in the trichomoniasis pilot vaccine trial, V=vaccinated and C=control / unvaccinated. Group Age Challenge Post Challenge 1 Challenge Post Challenge 2 1 2 Date 6 / 6 13 / 6 20 / 6 30 / 6 13 / 7 29 / 7 11 / 8 25 / 8 23 / 9 29 / 9 13 / 10 3 / 11 V 6 Neg Pos Neg Neg Neg Neg Neg Neg Neg Pos Pos Pos V 7 Neg Neg Neg Pos Neg Neg Neg Neg Neg Neg Pos Pos V 5 Neg Pos (Pos) Pos Pos Pos Pos Pos (Pos)* Pos Pos Pos V 4 Neg Neg Neg Neg Neg Neg Neg Neg Neg (Pos) Neg Neg V 5 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg V 6 Neg Pos (Pos) Pos Pos Pos Pos Pos Pos* (Pos) Neg Pos C 5 Neg Pos Pos Neg Pos Pos Pos Pos Pos* Pos Pos Pos C 7 Neg Neg Neg Pos Neg Pos Pos Pos Neg* Neg Neg Neg C 5 Neg Neg Neg Pos Neg Pos Neg Neg Neg Neg Pos Pos C 5 Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Pos Pos C 8 Neg Pos Pos Neg Neg Neg Neg Neg Neg Neg (Pos) Neg C 6 Neg Pos Pos Pos Pos Pos Pos Pos Pos* Pos Pos Pos *Not re-challenged / only challenged once on 6 / 6; (Pos) = low positive

[0210] For the 10 qPCR positive bulls selected for ‘treatment’ using two vaccine doses, four bulls tested persistently negative from the day of the first treatment which precluded these bulls from this experiment as they may have self-cured (highlighted in Table 4). A fifth bull although negative on the day of treatment was positive on all other sampling days. Of the remaining 5 bulls, four remained persistently positive, while one appeared to have cured after the 2nddose (highlighted in bold in Table 4). Thus essentially, out of 6 bulls (discounting the suspected four that ‘self-cured’ prior to dosing) one appeared to have cured through the treatments which is a 16.7% success rate. Table 4. Quantitative PCR results from the treated bulls (two doses of killed T. foetus TfOz5). Bulls testing consistently negative (self-cure) prior and post treatment are highlighted, and a treated bull in bold. Years 24 / 03 21 / 04 05 / 05 23 / 05 06 / 06 30 / 6 11 / 8 6 / 12 of age 1stDose 2ndDose 6 Pos Pos Pos Pos Pos Pos Pos Pos 7 Pos Pos Pos Pos Pos Pos Pos Pos 5 Pos Neg Neg Neg Neg Pos Neg Neg 4 Pos Pos Pos Pos Pos Pos Pos Pos 7 Pos Neg Neg Neg Neg Neg Neg Neg 7 Pos Neg Pos Pos Pos Pos Pos (Pos) 6 Pos Neg Neg Neg Neg Neg Neg (Pos) 6 Pos Neg Neg Neg Neg Neg Neg Neg 6 Pos Pos Pos Pos Neg Pox Pos Pos 4 Pos Pos Pos Pos Neg Neg Neg (Pos) Legend: (Pos) = low positiveExample 3. Media optimization for anaerobic fermentation and scale-up production Media optimization

[0211] Media was prepared with and without carbon source as described in Table 5 in a serum bottle. Culture from the second passage was used to inoculate the media of interest. The inoculation ratio was 0.8% (v / v). The culture was incubated at 30°C, 75 rpm for 96 hours (4 days). Each condition was conducted in single replicate. Cell density was measured every 24 hours with improved Neubauer cell counting instruments, the four large squares placed at the corners were used.

[0212] Similar to the carbon source selection studies described above, media with 20% and 40% (v / v) of inactivate serum was prepared as described in Table 6. No antibiotic was added. Culture from the second passage was used to inoculate the media of interest. The inoculation ratio was 0.8% (v / v). The culture was incubated at 30°C, 75 rpm for 144 hours (6 days). Each condition was conducted in two replicates. Cell density was measured every 24 hours with improved Neubauer cell counting instruments, the four large squares placed at the corners were used. Table 5. Media composition for carbon source selection Component Media 1 (control) Media 2 Media 3 TFM 2X 14 mL 13 mL 13 mL Antibiotic 1 mL 1 mL 1 mL Glucose (500 g / L) - 1 mL (final conc. 20 - g / L) Maltose (500 g / L) - - 1 mL (final conc. 20 g / L) Inactivate serum 10 mL 10 mL 10 mL Total volume 25 mL 25 mL 25 mL Table 6. Media composition with 20% and 40% (v / v) of inactivate serum Component Media with 20% Media with 40% TFM 2X 20 mL 15 mL Antibiotic - - Maltose (500 g / L) 0.8 mL (final conc.16 g / L) 0.6 mL (final conc.12 g / L) Inactivate serum 5 mL 10 mL Total volume 25 mL 25 mL

[0213] The cultivation of TfOZ5 and TfOz-N36 in TFM medium (without agar) in serum bottles achieved an average cell density of 7 × 10⁶ cells / mL. In the presence of glucose and maltose (20 g / L), both strains reached an average peak cell density of 1.1 × 10⁷ cells / mL. No significant difference in growth rate was observed between the strains when using maltose. However, TfOZ5 exhibited a higher growth rate than TfOz-N36 when cultured with glucose, reaching its peak at 48 hours, whereas TfOz-N36 peaked at 72 hours.

[0214] Two combinations of bovine serum (BVS) and maltose concentrations were tested to evaluate cell growth. Condition 1 contained 40% v / v BVS and 12 g / L maltose, while Condition 2 contained 20% v / v BVS and 16 g / L maltose. Both strains followed similar growth trends, but cells grew faster in Condition 2, suggesting that maltose concentration influences the growth rate. Cultures reached their peak cell concentrations after 72–96 hours, with strain-dependent maxima: 6 × 10⁶ cells / mL for TfOz-N36 and 9 × 10⁶ cells / mL for TfOz5. For scaling-up production, reducing BVS concentration and substituting it with maltose may be a viable alternative.

[0215] Media optimization for anaerobic fermentation in serum bottles and the scale-up production of protozoa TfOz-N36 and TfOZ5 successfully demonstrated scalability from serum bottles to 1 L and 15 L bioreactors (with a working volume of 8 L). Harvesting Protocol

[0216] For TfOz5 production, cells were harvested by centrifugation at 1,000 × g for 10 minutes at 4°C. The pellet was then washed in cold, sterile 1X PBS, with centrifugation at 1,000 × g for 10 minutes at 4°C between washes. By reducing the resuspension volume of 1X PBS, the cells were concentrated approximately 10 times. Cells were counted, and the volume was adjusted to achieve a final concentration of approximately 7.00 × 10⁷ cells / mL.

[0217] For TfOz-N36 production, cells were harvested by centrifugation at 1,000 × g for 10 minutes at 23°C (room temperature). The pellet was then washed in sterile 1X PBS, with centrifugation at 1,000 × g for 10 minutes at 23°C between washes. The resuspension volume of 1X PBS was reduced to obtain a 10x cell suspension. Cells were counted, and the volume was adjusted to achieve final concentrations of 1.37 x 107cells / mL for challenge 1 and 2.7 x 107cells / mL for challenge 2. Example 4. Large scale vaccination and treatment trial Bull vaccine trial

[0218] The trial consists of two groups (n=30 per group): a non-immunised (control) group and a vaccine group. Both groups were confirmed to be T. foetus negative ahead of the trial using qPCR. The vaccine group received two doses of the vaccine administered subcutaneously comprising the T. foetus TfOz5 strain (2 mL with 5 x 107cells per dose) on Day 0 and Day 27. The control group received two doses of mock PBS adjuvant injections subcutaneously on Day 0 and Day 27.

[0219] All bulls in both vaccine and control groups were challenged intrapreputially (i.e., in the foreskin of the penis) with the live T. foetus TfOz-N36 strain on Day 41 (challenge 1: 4.1 x 107live cells per dose) and Day 69 (challenge 2: 8.0 x 107live cells per dose). Blood and preputial samples were collected at multiple time points throughout the trial as indicated by arrows, ending on Day 189 (Figure 3). The presence or absence of T. foetus was subsequently confirmed using direct T. foetus qPCR and culture followed by microscopy and qPCR.

[0220] The inventors found that clearance of T. foetus is faster in vaccinated bulls than in unvaccinated bulls. As shown in Table 7 and Figure 4 a proportion of both vaccinated and unvaccinated control bulls were not positive after challenge potentially demonstrating natural resistance to trichomonosis in these controls. Nineteen of 28 bulls (two control bulls were removed from the trial) were positive in the control group while only 11 of 30 vaccinated bulls were transiently positive after the two live challenges. Figures 4 and 5 illustrate the efficacy of the T. foetus TfOz5 strain vaccine. Table 7. Number of infected bulls throughout the trial in the vaccine and the non-immunised group (Control). Days post first vaccination Control Vaccine 0 (first dose) 0 0 14 0 0 27 (second dose) 0 0 41 (first challenge) 0 0 55 6 3 69 (second challenge) 16 5 83 19 5 97 4 2 111 1 1 121 0 0 146 3 2 168 2 1 189 0 0 References

[0221] Abdel-Glil, M.Y., Solle, J., Wibberg, D. Neubauer, H. and Sprague, L.D. (2024) Chromosome-level genome assembly of Tritrichomonas foetus, the causative agent of Bovine Trichomonosis. Sci Data 11: 1030. https: / / doi.org / 10.1038 / s41597-024-03818-8

[0222] Benchimol, M., de Almeida, L.G.P., Vasconcelos, A.T., de Andrade Rosa, I., Reis Bogo, M., Kist, L.W. and de Souza, W. (2017) Draft genome sequence of Tritrichomonas foetus strain K. Genome Announcements 5 (16):e00195-17.

[0223] Buller, N., and Corney, B. (2013) Bovine trichomoniasis. Australian and New Zealand Standard Diagnostic Procedure, July 2013. ANZSDP-Bovine-trichomoniasis.pdf (awe.gov.au)

[0224] Carlton, J.M., Hirt, R.P., Silva, J.C., Delcher, A.L, Schatz, M., Zhao, Q., Wortman, J.R., Bidwell, S.L., Alsmark, U.C.M., Bestiero, S. et al. (2007) Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315(5809):207-212.

[0225] Clark, B.L., Dufty, J.H., and Parsonson I.M. (1983) Immunisation of bulls against trichomoniasis. Australian Veterinary Journal 60: 178-179.

[0226] Clark, B.L., Emery, D.L., and Dufty J.H. (1984) Therapeutic immunisation of bulls with the membranes and glycoproteins of Tritrichomonas foetus var. brisbane. Australian Veterinary Journal 61:65-66.

[0227] Edmondson MA, Joiner KS, Spencer JA, Riddell KP, Rodning SP, Gard JA, et al. Impact of a killed Tritrichomonas foetus vaccine on clearance of the organism and subsequent fertility of heifers following experimental inoculation. Theriogenology. (2017) 90:245–51. doi: 10.1016 / j.theriogenology.2016.09.056.

[0228] Fuchs, L.I., Fort, M.C., Cano, D, Bonetti, C.M., Giménez H.D., Vázquez, P.M., Bacigalupe, D, Breccia, J.D., Campero, C.M. amd Oyhenart, J.A. Clearance of Tritrichomonas foetus in experimentally infected heifers protected with vaccines based on killed-T. foetus with different adjuvants. Vaccine (2017).35.10.1016 / j.vaccine.2016.12.030.

[0229] Green, E. (2001) Strategies for the systematic sequencing of complex genomes. Nature Reviews Genetics 2: 573–583.

[0230] Hall, M., Wallace, B., Acree, W.M., and Chavez L.G. (1989) Bovine vaccine compositions and method for preventing trichomonas infections using same. US Patent US005223253A, June

[0231] Herr S, Ribeiro LM, Claassen E, Myburgh JG. A reduction in the duration of infection with Tritrichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls. Onderstepoort J Vet Res. (1991) 58:41–5.

[0232] Honigberg, B.M., and Mohn, F.A. (1973) An improved method for the isolation of highly polymerized native deoxyribonucleic acid from certain protozoa. Journal of Protozoology 20:146- 150.

[0233] Hudson D, Ball L, Cheney J, Mortimer R, Bowen R, Marsh D, et al. Testing of trichomoniasis vaccine in heifers mated to infected bulls. Theriogenology. (1993) 39:937–43. doi: 10.1016 / 0093-691X(93)90431-4

[0234] Irons, P.C., McGowan, M., de Assis, P.M., Randhawa, I., Awawdeh, L., Mugwabana, J., Barnes, T.S., Boe-Hansen, G., McCosker, K., Fordyce, G. (2022) Prevalence of Tritrichomonas foetus in beef bulls slaughtered at two abattoirs in northern Australia. Australian Veterinary Journal 100:201–204, doi: 10.1111 / avj.13149

[0235] Kvasnicka WG, Taylor RE, Huang JC, Hanks D, Tronstad RJ, Bosomworth A, et al. Investigations of the incidence of bovine trichomoniasis in nevada and of the efficacy of immunizing cattle with vaccines containing Tritrichomonas foetus. Theriogenology. (1989) 31:963–71. doi: 10.1016 / 0093-691X(89)90479-2

[0236] Kvasnicka WG, Hanks D, Huang JC, Hall MR, Sandblom D, Chu HJ, et al. Clinical evaluation of the efficacy of inoculating cattle with a vaccine containing Tritrichomonas foetus. Am J Vet Res. (1992) 53:2023–27.

[0237] Plastridge, W., and Williams, L.F. (1943) Observations on Vibrio fetus infection in cattle. Journal of the American Veterinary Medicine Association 102: 89-95.

[0238] McMillen, L., and Lew, A.E. (2006) Improved detection of Tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time PCR assay. Veterinary Parasitology 141: 204-215

[0239] Riley, D.E., and Krieger, J.N. (1992) Rapid and practical DNA isolation from Trichomonas vaginalis and other nuclease-rich protozoa. Molecular and Biochemical Parasitology 51:161-163.

[0240] Slapeta, J., Müller, N., Stack, C.M., Walker, G., Lew-Tabor, A., Tachezy, J., and Frey, C.F. (2012) Comparative analysis of Tritrichomonas foetus (Riedmüller, 1928) cat genotype, T. foetus (Riedmüller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci. International Journal for Parasitology 42(13-14):1143-1149.

[0241] Sutherland, A.K., Simmonds, G.C. and Bell, A.T. (1953) An outbreak of bovine trichomoniasis. Australian Veterinary Journal 29:67-69.

[0242] Tabor, A.E., Boe-Hansen, G., Hayes, B., Turni, C., Blackall, P., Ong, C.T., Fordyce, G., Forutan, M., Juli, S.B., Pious, P., Muller J., Diallo, I. (2021) Improving fertility in northern cattle through host and pathogen molecular diagnosis. MLA Donor Company project P.PSH.0799 Milestone 7 Report. October 2021.

[0243] Wang, A.L., and Wang C.C. (1992) Isolation and characterization of DNA from Tritrichomonas foetus and Trichomonas vaginalis. Molecular and Biochemical Parasitology 14: 323-335.

Claims

CLAIMS:

1. An immunogenic composition comprising a protozoal strain of the species Tritrichomonas foetus having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof and optionally one or more pharmaceutically acceptable carriers, diluents or excipients.

2. The immunogenic composition of claim 1, wherein the protozoal strain has a genomic sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

3. The immunogenic composition of claim 1 or 2, wherein the protozoal strain has the genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof.

4. The immunogenic composition of any one of claims 1 to 3, wherein the protozoal strain is a Tritrichomonas foetus strain Oz5 or a Tritrichomonas foetus strain Oz-N36.

5. The immunogenic composition of any one of claims 1 to 4, wherein the protozoal strain is a Tritrichomonas foetus strain Oz5 having ATCC Accession Number PTA-127585 or a Tritrichomonas foetus strain Oz-N36 having ATCC Accession Number PTA-127586.

6. The immunogenic composition of any one of claims 1 to 5, wherein the protozoal strain is inactivated or attenuated, optionally wherein the protozoal strain is inactivated by any one of pasteurisation, acidic pH, solvent / detergent treatment, ultraviolet light and combinations thereof.

7. The immunogenic composition of any one of claims 1 to 6, comprising about 1 x 104to about 1 x 109of the protozoal strain per unit dose.

8. The immunogenic composition of any one of claims 1 to 7, further comprising an adjuvant, optionally wherein the adjuvant is or comprises a water-in-oil emulsion.

9. The immunogenic composition of claim 8, wherein the adjuvant is a Montanide adjuvant.

10. An isolated Tritrichomonas foetus strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotidesequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof, or having ATCC Accession Number PTA-127585, or ATCC Accession Number PTA-127586.

11. An isolated cell or isolated population of cells of a Tritrichomonas foetus strain having a genomic sequence comprising or assembled from: nucleotide sequences set forth in SEQ ID NOs: 1 to 194, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 195 to 562, or a variant thereof, or nucleotide sequences set forth in SEQ ID NOs: 563 to 756, or a variant thereof, or having ATCC Accession Number PTA-127585, or ATCC Accession Number PTA-127586.

12. A method of producing the protozoal strain of any one of claims 1 to 9, said method including the step of culturing one or more cells of the protozoal strain in a cell culture medium.

13. The method of claim 12, further including the step of at least partially isolating the one or more cells from the cell culture medium and / or the step of inactivating or attenuating the protozoal strain.

14. The method of claim 12 or 13, wherein the cell culture medium is or comprises Tritrichomonas foetus medium (TFM) and / or is a liquid cell culture medium.

15. The method of any one of claims 12 to 14, wherein the cell culture medium further comprises at least one antimicrobial agent selected from the group consisting of an antibacterial, an antifungal, an antimycotic and combinations thereof, optionally wherein the at least one antimicrobial agent is selected from the group consisting of amphotericin B, penicillin, streptomycin, gentamicin, tetracycline, normacin, nystatin and combinations thereof.

16. A method of eliciting an immune response in a mammal, the method comprising administering a first dose comprising a therapeutically effective amount of the immunogenic composition of any one of claims 1 to 9 to the mammal to thereby elicit an immune response.

17. A method of treating, preventing or ameliorating a protozoal infection in a mammal, the method comprising administering a first dose comprising a therapeutically effective amount of the immunogenic composition of any one of claims 1 to 9 to the mammal to thereby treat, prevent or ameliorate the protozoal infection.

18. The method of claim 16 or 17, the method further including the step of administering a second dose comprising a therapeutically effective amount of the immunogenic composition of any one of claims 1 to 11 after the first dose.

19. The method of claim 18, wherein the second dose is administered to the mammal at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 days after administration of the first dose.

20. The method of any one of claims 16 to 19, wherein the immunogenic composition is administered parenterally

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